Difference between revisions of "Team:Freiburg/Notebook"

 
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div.panel {
 
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    padding: 0 18px;
 
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</style>
 
</style>
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</head>
 
</head>
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<body class="skin-vector">
 
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<div id="page-container">
 
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<div id="page-base" class="noprint"></div>
 
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<div id="head-base" class="noprint"></div>
 
  
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<!-- start div id=content -->
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<body>
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<div id="content">
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  <a name="top" id="top"></a>
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  <a name="dokuwiki__top" id="dokuwiki__top"></a>
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  <!-- start main content area -->
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  <!-- start div id bodyContent -->
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    <div style="padding-top: 80px; padding-bottom: 80px;">
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  <div id="bodyContent" class="dokuwiki">
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    <!-- start rendered wiki content -->
+
  
 +
    <h1 align="center"><a href="https://2017.igem.org/Team:Freiburg/Notebook_Cell_culture">Lab Notebook Cell Culture</a></h1>
 +
    <h1 align="center"><a href="https://2017.igem.org/Team:Freiburg/Notebook_Modeling">Lab Notebook Modeling</a></h1>
 +
    <h1 align="center"><a href="https://2017.igem.org/Team:Freiburg/Notebook_Cloning">Lab Notebook Cloning</a></h1>
 +
    <h1 align="center"><a href="https://2017.igem.org/Team:Freiburg/Notebook_Knockout">Lab Notebook Knockout</a></h1>
 +
    <h1 align="center"><a href="https://2017.igem.org/Team:Freiburg/Notebook_Suicide_Gene">Lab Notebook Suicide Gene</a></h1>
  
−
<div class="container">
+
     </div>
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     <div class="row">
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−
      <div class="col-md-12 text-center">
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−
        <div class="flex-container">
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          <div class="item">
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−
<h1 class="sectionedit1" id="lab_notebook_cell_culture">Lab Notebook Cell Culture</h1>
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−
<div class="level1">
+
−
 
+
−
</div>
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−
 
+
−
             
+
−
              <button class="accordion">April</button>
+
−
        <div id="vanish" class="panel panel-default">
+
−
<h3 class="sectionedit3" id="pei_transfection_of_hek_cells">PEI transfection of HEK cells</h3>
+
−
<div class="level3">
+
−
<ol>
+
−
<li class="level1"><div class="li"> Split the cells 1:5 into 10 cm plate (on 21.04.17)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Thaw PEI reagent at RT</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Dilute plasmid in 1 ml serum free DMEM (2.5µg from each plasmid)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add 15 µl PEI(1µg/µl) to the mix while vortexing (PEI:DNA=3:1)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> incubate 15 min at RT</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add the mix to the culture</div>
+
−
</li>
+
−
</ol>
+
−
 
+
−
</div>
+
−
</div>
+
−
 
+
−
<button class="accordion">May</button>
+
−
        <div id="vanish" class="panel panel-default">
+
−
<h2 class="sectionedit4" id="section130517">13.05.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
 
+
−
<h3 class="sectionedit5" id="pei_transfection_of_hek_jurkat_hpb-all_and_hut78_cells">PEI transfection of HEK, Jurkat, HPB-ALL and HUT78 cells</h3>
+
−
<div class="level3">
+
−
<ol>
+
−
<li class="level1"><div class="li"> Split cells 1:5 to a 6-well plate: 100 µl/well (on 12.05.17)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> For each sample: 3 µg plasmid DNA + 200 µl serum free DMEM</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Establish Mastermix</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add 9 µg PEI to each mix while vortexing (PEI:DNA=3:1)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 15 min incubation</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add the Mix to the cells: 275.25 µl GFP-Mix or 215.5 µl to corresponding samples</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> After 48h: Fluorescence Microscopy</div>
+
−
</li>
+
−
</ol>
+
−
<div class="table sectionedit6"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Plasmid number</th><th class="col1">component</th><th class="col2">concentration[ng/µL]</th><th class="col3">volume[µL] for 12 µg</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">pIG17_008</td><td class="col1"><strong>CMV_mCherry-delta-PstI_pA</strong></td><td class="col2">471.5</td><td class="col3">25</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">pIG17_009</td><td class="col1"><strong>CMV_EGFP_pA</strong></td><td class="col2">368.4</td><td class="col3">30</td>
+
−
</tr>
+
−
</table></div>
+
−
 
+
−
<h2 class="sectionedit7" id="section230517">23.05.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
 
+
−
<h3 class="sectionedit8" id="pei_transfection_of_hek_jurkat_hpb-all_and_hut78_cells1">PEI transfection of HEK, Jurkat, HPB-ALL and HUT78 cells</h3>
+
−
<div class="level3">
+
−
<div class="table sectionedit9"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Plasmid number</th><th class="col1">component</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">pIG17_008</td><td class="col1"><strong>CMV_mCherry-delta-PstI_pA</strong></td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">pIG17_009</td><td class="col1"><strong>CMV_EGFP_pA</strong></td>
+
−
</tr>
+
−
</table></div>
+
−
<ol>
+
−
<li class="level1"><div class="li"> Split cells 1:5 to a 6-well plate: 330 µl (from 10 cm plate)/well (22.05.17)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> For each sample: 3 µg plasmid DNA + 200 µl serum free DMEM</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Establish Mastermix</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add PEI to each mix while vortexing (PEI:DNA=3:1)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 15 min incubation</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add the Mix to the cells</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> After 48h: Fluorescence Microscopy &amp; Flow Cytometry</div>
+
−
</li>
+
−
</ol>
+
−
 
+
−
</div>
+
−
 
+
−
<h3 class="sectionedit10" id="lipotransfection_of_jurkat_hpb-all_and_hut78_cells">Lipotransfection of Jurkat, HPB-ALL and HUT78 cells</h3>
+
−
<div class="level3">
+
−
<div class="table sectionedit11"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Plasmid number</th><th class="col1">component</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">pIG17_009</td><td class="col1"><strong>CMV_EGFP_pA</strong></td>
+
−
</tr>
+
−
</table></div>
+
−
<ol>
+
−
<li class="level1"><div class="li"> pGFP: 2.5 µg/well</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Mix I: 150 µl serum free DMEM and 6 µl LTX reagent</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Mix II: 700 µl SERUM FREE DMEM + 2.5 µg pGFP + 14 µl PLUS reagent </div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Mix 150 µl Mix I with 150 µl Mix II</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 5 min incubation at RT</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add 300 µl to cells</div>
+
−
</li>
+
−
</ol>
+
−
 
+
−
</div>
+
−
 
+
−
<h2 class="sectionedit12" id="section300517">30.05.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
 
+
−
<h3 class="sectionedit13" id="biorad_electroporation">BioRad electroporation</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
Cell lines: HPB-ALL, HUT78 and Jurkat
+
−
Plasmid: pIG17_009 (GFP plasmid)
+
−
</p>
+
−
<ol>
+
−
<li class="level1"><div class="li"> Count the cells</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfer 1 mio. cells into falcons and centrifuge with 100g for 3 min</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Resuspend the cell pellet with 100 µl P3 Buffer</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add 3 µg pIG17_009</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfer the mixture into cuvette </div>
+
−
</li>
+
−
<li class="level1"><div class="li"> BioRad setting: 250 V, 960 µF, 35 sec.</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add 500 µl RPMI medium to the cells immediately after electroporation</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Plate the cells into 12-well plate with 2 ml Medium</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Over night culturing</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Wash the cuvettes for reusing</div>
+
−
</li>
+
−
</ol>
+
−
 
+
−
</div>
+
−
 
+
−
 
+
−
<h3 class="sectionedit15" id="preparation_for_lentiviral_transduction">preparation for lentiviral transduction</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
Plate 4 mio. Hek cells in 10 cm plate. (Will be around 7-8 mio cell the next day) <br/>
+
−
 
+
−
(optional: 5-6 mio. Hek cells in 15 cm plate)
+
−
</p>
+
−
</div>
+
−
</div>
+
−
</div>
+
−
 
+
−
<button class="accordion">June</button>
+
−
        <div id="vanish" class="panel panel-default">
+
−
<h2 class="sectionedit16" id="section060617">06.06.17</h2>
+
−
<div class="level2">
+
−
 
+
−
 
+
−
<h3 class="sectionedit17" id="production_of_lentivirus">Production of Lentivirus</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
Transfection of HEK cells for virus production.
+
−
</p>
+
−
<div class="table sectionedit18"><table class="inline">
+
−
<tr class="row0">
+
−
<td class="col0"><strong>Lentiviral packaging cells</strong></td><td class="col1">Hek293T cells</td>
+
−
</tr>
+
−
<tr class="row1">
+
−
<td class="col0"><strong>Plate size</strong></td><td class="col1">10 cm</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0"><strong>Total DNA/plate</strong></td><td class="col1">12.87 µg</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0"><strong>Transfer: gag/pol: env ratio</strong></td><td class="col1">2:1:1</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0"><strong>Construct</strong></td><td class="col1">6.43 µg</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0"><strong>pCMV∆R8.74</strong></td><td class="col1">3.22 µg</td>
+
−
</tr>
+
−
<tr class="row6">
+
−
<td class="col0"><strong>pMD2G</strong></td><td class="col1">3.22 µg</td>
+
−
</tr>
+
−
<tr class="row7">
+
−
<td class="col0"><strong>N/P ration</strong></td><td class="col1">15</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_18'></div><div class="table sectionedit19"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Plasmid number</th><th class="col1">component</th><th class="col2">concentration[ng/µL]</th><th class="col3">volume [µL]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">pIG17_082</td><td class="col1"><strong>Transfer plasmid with GFP p526</strong></td><td class="col2">253</td><td class="col3">25.4</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">pIG17_003</td><td class="col1"><strong>Envelope plasmid pMD2G</strong></td><td class="col2">105</td><td class="col3">30.7</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">pIG17_004</td><td class="col1"><strong>Packaging Plasmid pCMV∆R8.74</strong></td><td class="col2">150</td><td class="col3">21.5</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_19'></div><ul>
+
−
<li class="level1"><div class="li"> Packaging Mastermix</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit20"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Component</th><th class="col1">1x [µL]</th><th class="col2">2.1x [µL]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">pIG17_003</td><td class="col1">30.63</td><td class="col2">45.03</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">pIG17_004</td><td class="col1">21.44</td><td class="col2">64.33</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">150mM NaCl</td><td class="col1">441.49</td><td class="col2">927.12</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">total</td><td class="col1">493.56</td><td class="col2">1036.476</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_20'></div><ul>
+
−
<li class="level1"><div class="li"> PEI Mastermix</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit21"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Component</th><th class="col1">1x [µL]</th><th class="col2">2.1x [µL]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">23.2mM (1 µg/µl)PEI</td><td class="col1">24.94</td><td class="col2">52.41</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">150mM NaCl</td><td class="col1">475.04</td><td class="col2">997.59</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">total</td><td class="col1">499.98</td><td class="col2">1050</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_21'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_17'></div>
+
−
<h2 class="sectionedit22" id="section070617">07.06.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_22'></div>
+
−
<h3 class="sectionedit23" id="amaxa_electroporation">Amaxa electroporation</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
Cell lines: HUT78 and Jurkat
+
−
Plasmid: pIG17_008 (mCherry plasmid)
+
−
</p>
+
−
<ol>
+
−
<li class="level1"><div class="li"> Count the cells</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfer 1 mio. cells into falcons and centrifuge with 100g for 3 min</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Resuspend the cell pellet with 100 µl P3 Buffer</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add 3 µg pIG17_008</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfer the mixture into cuvette </div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Amaxa Programm: X-005</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add 500 µl RPMI medium to the cells immediately after electroporation</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Plate the cells into 12-well plate with 2 ml Medium</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Over night culturing</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Wash the cuvettes for reusing</div>
+
−
</li>
+
−
</ol>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_23'></div>
+
−
<h3 class="sectionedit24" id="production_of_lentivirus1">Production of Lentivirus</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Collect the medium (containing virus supernatant) and add new medium after 24h</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_24'></div>
+
−
<h3 class="sectionedit25" id="biorad_electroporation1">BioRad electroporation</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
Cell lines: HUT78 and Jurkat
+
−
Plasmid: pIG17_009 (GFP plasmid)
+
−
</p>
+
−
<ol>
+
−
<li class="level1"><div class="li"> Count the cells</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfer 1 mio. cells into falcons and centrifuge with 100g for 3 min</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Resuspend the cell pellet with 100 µl P3 Buffer</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add 3 µg pIG17_009</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfer the mixture into cuvette </div>
+
−
</li>
+
−
<li class="level1"><div class="li"> BioRad setting: 250 V, 960 µF, 35 sec.</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add 500 µl RPMI medium to the cells immediately after electroporation</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Plate the cells into 12-well plate with 2 ml Medium</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Over night culturing</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Wash the cuvettes for reusing</div>
+
−
</li>
+
−
</ol>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_25'></div>
+
−
<h2 class="sectionedit26" id="section080617">08.06.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_26'></div>
+
−
<h3 class="sectionedit27" id="production_of_lentivirus2">Production of Lentivirus</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Collect the medium (containing virus supernatant) and add new medium after 48h</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Mix with medium collected after 24h</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_27'></div>
+
−
<h3 class="sectionedit28" id="flow_cytometry">Flow Cytometry</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> negative controls: Hut78 and Jurkat cells.</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> positive control: GFP_Jurkat(p526)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Amaxa Samples: Hut78 (mCherry and GFP) and Jurkat (mCherry)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> BioRad Samples: Hut78 (GFP) and Jurkat (GFP)</div>
+
−
</li>
+
−
</ul>
+
−
<ol>
+
−
<li class="level1"><div class="li"> Count the cells</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> resuspend cell pellet (about 1 mio.) with FACS Buffer (PBS with 1%FCS)</div>
+
−
</li>
+
−
</ol>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_28'></div>
+
−
<h2 class="sectionedit29" id="section090617">09.06.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_29'></div>
+
−
<h3 class="sectionedit30" id="lentiviral_transduction">Lentiviral transduction</h3>
+
−
<div class="level3">
+
−
<ol>
+
−
<li class="level1"><div class="li"> Plate about 200000 cells for each wells.</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Incubate the plates over weekend in incubator.</div>
+
−
</li>
+
−
</ol>
+
−
<div class="table sectionedit31"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Jurkat sample</th><th class="col1">Medium [ml]</th><th class="col2">Virus [ml]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1</td><td class="col1">1</td><td class="col2">1</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2</td><td class="col1">0</td><td class="col2">2</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_31'></div><div class="table sectionedit32"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Hut78 sample</th><th class="col1">Medium [ml]</th><th class="col2">Virus [ml]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1</td><td class="col1">2</td><td class="col2">0</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2</td><td class="col1">1.5</td><td class="col2">0.5</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">3</td><td class="col1">1</td><td class="col2">1</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">4</td><td class="col1">0</td><td class="col2">2</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_32'></div><div class="table sectionedit33"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">HEK sample</th><th class="col1">Medium [ml]</th><th class="col2">Virus [ml]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1</td><td class="col1">2</td><td class="col2">0</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2</td><td class="col1">1.5</td><td class="col2">0.5</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">3</td><td class="col1">1</td><td class="col2">1</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">4</td><td class="col1">0</td><td class="col2">2</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_33'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_30'></div>
+
−
<h2 class="sectionedit34" id="section100617">10.06.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_34'></div>
+
−
<h3 class="sectionedit35" id="biorad_electroporation2">BioRad electroporation</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Cell lines: Hut78</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Plasmid: pIG17_008 (mCherry plasmid)</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit36"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Component</th><th class="col1">Amount</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">Hut cells</td><td class="col1">3 mio. cells</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">pIG17_008</td><td class="col1">5 µg</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_36'></div><div class="table sectionedit37"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Setting</th><th class="col1">Capacitance[µF]</th><th class="col2">Voltage</th><th class="col3">Cuvette size[cm]</th><th class="col4">Time Constance [msec]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">Setting 1</td><td class="col1">250</td><td class="col2">150</td><td class="col3">0.2</td><td class="col4">4.0</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">Setting 2</td><td class="col1">500</td><td class="col2">120</td><td class="col3">0.2</td><td class="col4">10.1</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_37'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_35'></div>
+
−
<h2 class="sectionedit38" id="section120617">12.06.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_38'></div>
+
−
<h3 class="sectionedit39" id="lentiviral_transduction1">Lentiviral transduction</h3>
+
−
<div class="level3">
+
−
<ol>
+
−
<li class="level1"><div class="li"> After 72h → Change the medium of the plates (for suspension cells: first centrifuge and then resuspend cell pellet with new medium)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Incubation over night for FACS</div>
+
−
</li>
+
−
</ol>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_39'></div>
+
−
<h3 class="sectionedit40" id="flow_cytometry_for_biorad_electroporation_on_june_10th">Flow Cytometry for BioRad electroporation on June 10th</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
no positive population
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_40'></div>
+
−
<h2 class="sectionedit41" id="section130617">13.06.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_41'></div>
+
−
<h3 class="sectionedit42" id="flow_cytometry_for_lentiviral_infected_cells">Flow Cytometry for lentiviral infected cells</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
with ration of 1:1 (Medium vs. Virus medium) cells were most frequently infected and viable.
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_42'></div>
+
−
<h3 class="sectionedit43" id="biorad_electroporation3">BioRad Electroporation</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Cell lines: Jurkat &amp; Hut78</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Plasmid: pIG17_009</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Cell count: 1 mio. (Jurkat) 4 mio. (Hut78)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Settings: </div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit44"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Cell type</th><th class="col1">Setting</th><th class="col2">Capacitance[µF]</th><th class="col3">Voltage</th><th class="col4">resistence</th><th class="col5">Time Constance [msec]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">Jurkat</td><td class="col1">Setting 1</td><td class="col2">300</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">35</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">Jurkat</td><td class="col1">Setting 2</td><td class="col2">350</td><td class="col3">200</td><td class="col4">1000Ω</td><td class="col5">35</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">Hut78</td><td class="col1">Setting 1</td><td class="col2">250</td><td class="col3">150</td><td class="col4">∞</td><td class="col5">50</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">Hut78</td><td class="col1">Setting 2</td><td class="col2">500</td><td class="col3">120</td><td class="col4">∞</td><td class="col5">50</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_44'></div>
+
−
</div>
+
−
 
+
−
 
+
−
 
+
−
<div class='secedit editbutton_section editbutton_43'></div>
+
−
<h2 class="sectionedit45" id="section140617">14.06.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_45'></div>
+
−
<h3 class="sectionedit46" id="amaxa_electroporation1">Amaxa electroporation</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Cell lines: Jurkat &amp; Hut78</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Plasmid: pIG17_009</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Cell count: 1 mio.</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Program: X-005</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Flow cytometry after 24h.</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_46'></div>
+
−
<h3 class="sectionedit47" id="flow_cytometry_for_biorad_electroporated_jk_cells">Flow Cytometry for BioRad electroporated JK cells</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
No positive cell population.
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_47'></div>
+
−
<h2 class="sectionedit48" id="section150617">15.06.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_48'></div>
+
−
<h3 class="sectionedit49" id="flow_cytometry_for_biorad_electroporated_hut78_cells_and_amaxa_electroporated_t_cells">Flow Cytometry for BioRad electroporated Hut78 cells and Amaxa electroporated T cells</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
no positive cell population.
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_49'></div>
+
−
<h2 class="sectionedit50" id="section160617">16.06.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_50'></div>
+
−
<h3 class="sectionedit51" id="mycoplasma_test_lentiviral_transduced_jurkat_and_hut78">Mycoplasma test(lentiviral transduced Jurkat and Hut78)</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Jurkat(p526): mycoplasma positive</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Hut78(p526):mycoplasma positive</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> HEK(p526): mycoplasma negative</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
<p>
+
−
No cell sorting, stock cultures should be sent for test.
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_51'></div>
+
−
<h2 class="sectionedit52" id="section170617">17.06.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_52'></div>
+
−
<h3 class="sectionedit53" id="biorad_electroporation_with_jk_cells">BioRad Electroporation with JK cells</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Cell lines: Jurkat</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Plasmid: pIG17_009</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Cell count: 2 mio./ approach</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 4 approaches: </div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit54"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Approach</th><th class="col1">Capacitance[µF]</th><th class="col2">Voltage</th><th class="col3">Cuvette size[cm]</th><th class="col4">Time Constance [msec]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1:negative control</td><td class="col1">960</td><td class="col2">250</td><td class="col3">0.2</td><td class="col4">35</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2:condition 1</td><td class="col1">960</td><td class="col2">250</td><td class="col3">0.2</td><td class="col4">35</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">3:condition 2</td><td class="col1">960</td><td class="col2">250</td><td class="col3">0.2</td><td class="col4">35</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">4:condition 3</td><td class="col1">960</td><td class="col2">250</td><td class="col3">0.2</td><td class="col4">40</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_54'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_53'></div>
+
−
<h3 class="sectionedit55" id="pei_transfection_with_hek_cells">PEI transfection with HEK cells</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
4 approaches: <br/>
+
−
 
+
−
</p>
+
−
<ol>
+
−
<li class="level1"><div class="li"> 1 transfection with pIG17_009</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 2 transfections with pIG17_013</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 1 untransfected as control</div>
+
−
</li>
+
−
</ol>
+
−
<ol>
+
−
<li class="level1"><div class="li"> Split cells 1:5 to a 6-well plate: 100 µl/well (on 16.06.17)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> For each sample: 3 µg plasmid DNA + 200 µl serum free DMEM</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Establish Mastermix</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add 9 µg PEI to each mix while vortexing (PEI:DNA=3:1)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 15 min incubation</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add the Mix to the cells</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> After 48h: Fluorescence Microscopy &amp; Flow cytometry</div>
+
−
</li>
+
−
</ol>
+
−
<div class="table sectionedit56"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Plasmid number</th><th class="col1">component</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">pIG17_013</td><td class="col1"><strong>pHRE_Ptal_EGFP_pA</strong></td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_56'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_55'></div>
+
−
<h2 class="sectionedit57" id="section190617">19.06.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_57'></div>
+
−
<h3 class="sectionedit58" id="hypoxia_test_cocl2">Hypoxia test (CoCl2)</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Cell: via PEI transfected HEK cells.<br/>
+
−
</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> CoCl2 stock: 1M<br/>
+
−
</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> End concentration of CoCl2: 100µM<br/>
+
−
</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Cell observation under fluorescence microscopy after 2h:<br/>
+
−
</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
<p>
+
−
-By adding CoCl2, the HRE-GFP transfected cells showed less fluorescence signal than without CoCl2…<br/>
+
−
 
+
−
</p>
+
−
<ul>
+
−
<li class="level1"><div class="li"> Flow cytometry: no expected signal</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_58'></div>
+
−
<h3 class="sectionedit59" id="biorad_electroporation4">BioRad Electroporation</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Cell lines: Jurkat</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Plasmid: pIG17_009</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Cell count: 2 mio./ approach</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Setting: 96 well plates, well set 1(ABCD1)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 4 approaches: </div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit60"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Approach</th><th class="col1">Capacitance[µF]</th><th class="col2">Voltage</th><th class="col3">Cuvette size[cm]</th><th class="col4">Time Constance [msec]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1:negative control</td><td class="col1">960</td><td class="col2">250</td><td class="col3">0.2</td><td class="col4">35</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2:condition1</td><td class="col1">960</td><td class="col2">250</td><td class="col3">0.2</td><td class="col4">35</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">3:condition2</td><td class="col1">960</td><td class="col2">250</td><td class="col3">0.2</td><td class="col4">30</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">4:condition3</td><td class="col1">960</td><td class="col2">250</td><td class="col3">0.2</td><td class="col4">40</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_60'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_59'></div>
+
−
<h2 class="sectionedit61" id="section200617">20.06.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_61'></div>
+
−
<h3 class="sectionedit62" id="biorad_electroporation5">BioRad electroporation</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Cell lines: Jurkat</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Plasmid: pIG17_009</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Cell count: 2 mio./ approach</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Setting: 96 well plates, well set 1(ABCD1), Expotential</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 4 approaches: </div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit63"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Approach</th><th class="col1">Capacitance[µF]</th><th class="col2">Voltage</th><th class="col3">Cuvette size[cm]</th><th class="col4">Resistance</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1:negative control</td><td class="col1">950</td><td class="col2">250</td><td class="col3">0.2</td><td class="col4">1000Ω</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2:condition1</td><td class="col1">350</td><td class="col2">250</td><td class="col3">0.2</td><td class="col4">∞</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">3:condition2</td><td class="col1">300</td><td class="col2">300</td><td class="col3">0.2</td><td class="col4">1000Ω</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">4:condition3</td><td class="col1">300</td><td class="col2">250</td><td class="col3">0.2</td><td class="col4">1000Ω</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_63'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_62'></div>
+
−
<h2 class="sectionedit64" id="section210617">21.06.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_64'></div>
+
−
<h3 class="sectionedit65" id="mycoplasma-test_stock_cultures">Mycoplasma-Test (Stock cultures)</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> HEK293T: mycoplasma negative</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Jurkat: mycoplasma negative</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Hut78: mycoplasma positive → abort culturing of Hut78!</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_65'></div>
+
−
<h3 class="sectionedit66" id="flow_cytometry_biorad_on_jun19th">Flow Cytometry (BioRad on Jun.19th)</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Not successful</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_66'></div>
+
−
<h2 class="sectionedit67" id="section240617">24.06.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_67'></div>
+
−
<h3 class="sectionedit68" id="pei_transfection_pig17_013_for_hypoxia_test">PEI transfection (pIG17_013) for hypoxia test</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> In total: 3*6 wells </div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfection with pIG17_009 as positive control: 1 well/plate</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfection with pIG17_013: 4 well/plate</div>
+
−
</li>
+
−
</ul>
+
−
<ol>
+
−
<li class="level1"><div class="li"> Split the cells 1:5 into 10 cm plate (on 23.06.17)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Thaw PEI reagent at RT</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Dilute plasmid in 1 ml serum free DMEM (3µg from each plasmid)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add 15 µl PEI(1µg/µl) to the mix while vortexing (PEI:DNA=3:1)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> incubate 15 min at RT</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add 200µl mix to wells</div>
+
−
</li>
+
−
</ol>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_68'></div>
+
−
<h2 class="sectionedit69" id="section250617">25.06.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_69'></div>
+
−
<h3 class="sectionedit70" id="hypoxia_test_cocl21">Hypoxia test (CoCl2)</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Cell: 1 plate of via PEI transfected HEK cells. (from 24.06)<br/>
+
−
</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> CoCl2 stock: 1M<br/>
+
−
</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> End concentration of CoCl2: 100µM<br/>
+
−
</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit71"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Well</th><th class="col1">End concentration of CoCl2[µM]</th><th class="col2">Volume of Stock CoCl2[µl]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1: untransfected</td><td class="col1">100</td><td class="col2">200</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2:pIG17_009 transfected</td><td class="col1">-</td><td class="col2">-</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">3:pIG17_013 transfected</td><td class="col1">-</td><td class="col2">-</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">4:pIG17_013 transfected</td><td class="col1">50</td><td class="col2">100</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">5:pIG17_013 transfected</td><td class="col1">100</td><td class="col2">200</td>
+
−
</tr>
+
−
<tr class="row6">
+
−
<td class="col0">6:pIG17_013 transfected</td><td class="col1">200</td><td class="col2">400</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_71'></div>
+
−
<p>
+
−
  * Cell observation under fluorescence microscopy every hour after CoCl2 treatment
+
−
</p>
+
−
<ul>
+
−
<li class="level1"><div class="li"> Flow cytometry after 8 hours</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_70'></div>
+
−
<h3 class="sectionedit72" id="biorad_electroporation6">BioRad Electroporation</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Cell lines: Jurkat</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Plasmid: pIG17_009</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Cell count: 2 mio./ approach</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Setting: 12 well plates, well set 1</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 3 approaches: </div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit73"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Approach</th><th class="col1">Capacitance[µF]</th><th class="col2">Voltage</th><th class="col3">Resistance</th><th class="col4">Time Constance [msec]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1:3 µg plasmid</td><td class="col1">960</td><td class="col2">250</td><td class="col3">∞</td><td class="col4">20</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2:6 µg plasmid</td><td class="col1">960</td><td class="col2">250</td><td class="col3">∞</td><td class="col4">20</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">3:9 µg plasmid</td><td class="col1">960</td><td class="col2">250</td><td class="col3">∞</td><td class="col4">20</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_73'></div><ul>
+
−
<li class="level1"><div class="li"> Flow cytometry after 24h. </div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_72'></div>
+
−
<h2 class="sectionedit74" id="section260617">26.06.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_74'></div>
+
−
<h3 class="sectionedit75" id="hypoxia_test_cocl22">Hypoxia test (CoCl2)</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
<strong>1.Plate</strong>
+
−
</p>
+
−
<div class="table sectionedit76"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Duration of Treatment</th><th class="col1">Well</th><th class="col2">End concentration of CoCl2[µM]</th><th class="col3">Volume of Stock CoCl2[µl]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">6h</td><td class="col1">1: untransfected</td><td class="col2">100</td><td class="col3">200</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">6h</td><td class="col1">2:pIG17_009 transfected</td><td class="col2">-</td><td class="col3">-</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">6h</td><td class="col1">3:pIG17_013 transfected</td><td class="col2">-</td><td class="col3">-</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">6h</td><td class="col1">4:pIG17_013 transfected</td><td class="col2">50</td><td class="col3">100</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">6h</td><td class="col1">5:pIG17_013 transfected</td><td class="col2">100</td><td class="col3">200</td>
+
−
</tr>
+
−
<tr class="row6">
+
−
<td class="col0">6h</td><td class="col1">6:pIG17_013 transfected</td><td class="col2">200</td><td class="col3">400</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_76'></div>
+
−
<p>
+
−
<strong>2.Plate</strong>
+
−
</p>
+
−
<div class="table sectionedit77"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Duration of Treatment</th><th class="col1">Well</th><th class="col2">End concentration of CoCl2[µM]</th><th class="col3">Volume of Stock CoCl2[µl]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">24h</td><td class="col1">1: untransfected</td><td class="col2">100</td><td class="col3">200</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">24h</td><td class="col1">2:pIG17_009 transfected</td><td class="col2">-</td><td class="col3">-</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">24h</td><td class="col1">3:pIG17_013 transfected</td><td class="col2">-</td><td class="col3">-</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">24h</td><td class="col1">4:pIG17_013 transfected</td><td class="col2">50</td><td class="col3">100</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">24h</td><td class="col1">5:pIG17_013 transfected</td><td class="col2">100</td><td class="col3">200</td>
+
−
</tr>
+
−
<tr class="row6">
+
−
<td class="col0">24h</td><td class="col1">6:pIG17_013 transfected</td><td class="col2">200</td><td class="col3">400</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_77'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_75'></div>
+
−
<h3 class="sectionedit78" id="flow_cytometry_hypoxia_test_biorad">Flow Cytometry (Hypoxia test &amp; BioRad)</h3>
+
−
<div class="level3">
+
−
 
+
−
</div>
+
−
 
+
−
<h4 id="hypoxia_test">Hypoxia test</h4>
+
−
<div class="level4">
+
−
 
+
−
<p>
+
−
<strong>No induction of GFP expression under hypoxia condition</strong>
+
−
<strong>Problems:</strong>
+
−
</p>
+
−
<ul>
+
−
<li class="level1"><div class="li"> Cells stayed attached to the ground of plates even after treating with trypsin</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Cells clumps instead of single cell were detected</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
 
+
−
<h4 id="biorad_electroporation7">BioRad Electroporation</h4>
+
−
<div class="level4">
+
−
 
+
−
<p>
+
−
By increasing the amount of plasmid DNA, some cells were potentially transfected!!!
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_78'></div>
+
−
<h2 class="sectionedit79" id="section270617">27.06.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_79'></div>
+
−
<h3 class="sectionedit80" id="biorad_electroporation8">BioRad Electroporation</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
<strong>9µg for each approach</strong><br/>
+
−
 
+
−
</p>
+
−
<ul>
+
−
<li class="level1"><div class="li"> Setting: 12 well plate, 1 well set, 1 pulse</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
 
+
−
<h4 id="gfp_plasmidpig17_009">GFP Plasmid: pIG17_009</h4>
+
−
<div class="level4">
+
−
<div class="table sectionedit81"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Approach</th><th class="col1">Capacitance[µF]</th><th class="col2">Voltage</th><th class="col3">Resistance</th><th class="col4">Time Constance [msec]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1</td><td class="col1">950</td><td class="col2">250</td><td class="col3">∞</td><td class="col4">20</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2</td><td class="col1">950</td><td class="col2">250</td><td class="col3">1000Ω</td><td class="col4">20</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">3</td><td class="col1">950</td><td class="col2">250</td><td class="col3">1500Ω</td><td class="col4">20</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_81'></div>
+
−
</div>
+
−
 
+
−
<h4 id="knock_out">Knock out</h4>
+
−
<div class="level4">
+
−
<div class="table sectionedit82"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Approach</th><th class="col1">Plasmid</th><th class="col2">Capacitance[µF]</th><th class="col3">Voltage</th><th class="col4">Resistance</th><th class="col5">Time Constance [msec]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1</td><td class="col1">203 (Cas9) + 104 (gRNA)</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2</td><td class="col1">203 (Cas9)+ scramble</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">3</td><td class="col1">sgRNA3 (gRNA_GFP)</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">4</td><td class="col1">sgRNA3 (gRNA_GFP)</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_82'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_80'></div>
+
−
<h2 class="sectionedit83" id="section280617">28.06.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_83'></div>
+
−
<h3 class="sectionedit84" id="flow_cytometryelectroporation_on_june_27th">Flow Cytometry: electroporation on June 27th</h3>
+
−
<div class="level3">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_84'></div>
+
−
<h2 class="sectionedit85" id="section290617">29.06.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_85'></div>
+
−
<h3 class="sectionedit86" id="production_of_lentivirus3">Production of Lentivirus</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
Transfection of HEK cells for virus production.
+
−
</p>
+
−
<div class="table sectionedit87"><table class="inline">
+
−
<tr class="row0">
+
−
<td class="col0"><strong>Lentiviral packaging cells</strong></td><td class="col1">Hek293T cells</td>
+
−
</tr>
+
−
<tr class="row1">
+
−
<td class="col0"><strong>Plate size</strong></td><td class="col1">10 cm</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0"><strong>Total DNA/plate</strong></td><td class="col1">12.87 µg</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0"><strong>Transfer: gag/pol: env ratio</strong></td><td class="col1">2:1:1</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0"><strong>Construct</strong></td><td class="col1">6.43 µg</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0"><strong>pCMV∆R8.74</strong></td><td class="col1">3.22 µg</td>
+
−
</tr>
+
−
<tr class="row6">
+
−
<td class="col0"><strong>pMD2G</strong></td><td class="col1">3.22 µg</td>
+
−
</tr>
+
−
<tr class="row7">
+
−
<td class="col0"><strong>N/P ration</strong></td><td class="col1">15</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_87'></div><div class="table sectionedit88"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Plasmid number</th><th class="col1">component</th><th class="col2">concentration[ng/µL]</th><th class="col3">volume [µL]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">pIG17_?</td><td class="col1"><strong>LentiCRISPR(AG Cathomen)</strong></td><td class="col2">1100</td><td class="col3">5.85</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">pIG17_003</td><td class="col1"><strong>Envelope plasmid pMD2G</strong></td><td class="col2">68</td><td class="col3">47.4</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">pIG17_004</td><td class="col1"><strong>Packaging Plasmid pCMV∆R8.74</strong></td><td class="col2">209</td><td class="col3">15.4</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_88'></div><ul>
+
−
<li class="level1"><div class="li"> Packaging Mastermix</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit89"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Component</th><th class="col1">1x [µL]</th><th class="col2">2.1x [µL]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">pIG17_003</td><td class="col1">47.4</td><td class="col2">99.54</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">pIG17_004</td><td class="col1">15.4</td><td class="col2">32.34</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">150mM NaCl</td><td class="col1">441.49</td><td class="col2">927.12</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">total</td><td class="col1">504.3</td><td class="col2">1059</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_89'></div><ul>
+
−
<li class="level1"><div class="li"> PEI Mastermix</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit90"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Component</th><th class="col1">1x [µL]</th><th class="col2">2.1x [µL]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">23.2mM (1µg/µl)PEI</td><td class="col1">24.94</td><td class="col2">52.41</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">150mM NaCl</td><td class="col1">475.04</td><td class="col2">997.59</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">total</td><td class="col1">499.98</td><td class="col2">1050</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_90'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_86'></div>
+
−
<h2 class="sectionedit91" id="section300617">30.06.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_91'></div>
+
−
<h3 class="sectionedit92" id="production_of_lentivirus4">Production of Lentivirus</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Collect the medium (containing virus supernatant) and add new medium after 24h</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
 
+
−
</div>
+
−
</div>
+
−
 
+
−
<button class="accordion">July</button>
+
−
        <div class="panel panel-default">
+
−
            <br>
+
−
                           
+
−
             
+
−
              <h2 class="sectionedit93" id="section010717">01.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_93'></div>
+
−
<h3 class="sectionedit94" id="production_of_lentivirus5">Production of Lentivirus</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Collect the medium (containing virus supernatant) and add new medium after 24h</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Mix the virus medium (24h + 48h)</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_94'></div>
+
−
<h3 class="sectionedit95" id="lentiviral_transduction2">Lentiviral transduction</h3>
+
−
<div class="level3">
+
−
<ol>
+
−
<li class="level1"><div class="li"> Plate about 200000 cells for each wells.</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Incubate the plates over weekend in incubator.</div>
+
−
</li>
+
−
</ol>
+
−
<div class="table sectionedit96"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Jurkat sample</th><th class="col1">Medium [ml]</th><th class="col2">Virus [ml]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1</td><td class="col1">2</td><td class="col2">0</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2</td><td class="col1">1.5</td><td class="col2">0.5</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">3</td><td class="col1">1</td><td class="col2">1</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">4</td><td class="col1">0.5</td><td class="col2">1.5</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">5</td><td class="col1">0</td><td class="col2">2</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_96'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_95'></div>
+
−
<h3 class="sectionedit97" id="pei_transfection_with_hek_for_hypoxia_test">PEI transfection with HEK for Hypoxia Test</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Prepare 2*6-well Plates of HEK cells</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfect cells via PEI:</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit98"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">well</th><th class="col1">plasmid</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1</td><td class="col1">-</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2</td><td class="col1">pIG17_009</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">3</td><td class="col1">pIG17_013</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">4</td><td class="col1">pIG17_013</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">5</td><td class="col1">pIG17_013</td>
+
−
</tr>
+
−
<tr class="row6">
+
−
<td class="col0">6</td><td class="col1">pIG17_013</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_98'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_97'></div>
+
−
<h3 class="sectionedit99" id="biorad_electroporationrepeat_pgfp_transfection">BioRad Electroporation: Repeat pGFP transfection</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
<strong>9µg for each approach</strong><br/>
+
−
 
+
−
</p>
+
−
<ul>
+
−
<li class="level1"><div class="li"> Setting: 12 well plate, 1 well set, 1 pulse</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit100"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Approach</th><th class="col1">Capacitance[µF]</th><th class="col2">Voltage</th><th class="col3">Resistance</th><th class="col4">Time Constance [msec]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1: no plasmid</td><td class="col1">950</td><td class="col2">250</td><td class="col3">∞</td><td class="col4">20</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2: Mock plasmid</td><td class="col1">950</td><td class="col2">250</td><td class="col3">∞</td><td class="col4">20</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">3: pIG17_009</td><td class="col1">950</td><td class="col2">250</td><td class="col3">∞</td><td class="col4">20</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">4: pIG17_009</td><td class="col1">950</td><td class="col2">250</td><td class="col3">1000Ω</td><td class="col4">20</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">5: pIG17_009</td><td class="col1">950</td><td class="col2">250</td><td class="col3">1500Ω</td><td class="col4">20</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_100'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_99'></div>
+
−
<h2 class="sectionedit101" id="section020717">02.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_101'></div>
+
−
<h3 class="sectionedit102" id="cocl2_treatment_with_jurkat_cells">CoCl2 treatment with Jurkat cells</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Cells treated with CoCl2 built clumps, which depends on the incubation time and CoCl2 concentrations. </div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_102'></div>
+
−
<h3 class="sectionedit103" id="repeat_hypoxia_test_24h_after_pei">Repeat Hypoxia test (24h after PEI)</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
<strong>No induction of GFP expression under hypoxia condition</strong>
+
−
</p>
+
−
<ul>
+
−
<li class="level1"><div class="li"> Add different concentrations of CoCl2 (50µM, 100µM and 200 µM)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Observe the cells under fluorescence microscope 3h, 6h, 8h, 16h, 18h and 24h after CoCl2 treatment</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> No detectable induction of gene expression for every approach</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
 
+
−
<h4 id="flow_cytometry_biorad_on_july_1st">Flow Cytometry (BioRad on July 1st)</h4>
+
−
<div class="level4">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Both negative and positive controls showed abnormal populations → need to repeat the experiments.</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_103'></div>
+
−
<h2 class="sectionedit104" id="section030717">03.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_104'></div>
+
−
<h3 class="sectionedit105" id="repeat_hypoxia_test_48h_after_pei">Repeat Hypoxia test (48h after PEI)</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
<strong>No induction of GFP expression under hypoxia condition</strong>
+
−
</p>
+
−
<ul>
+
−
<li class="level1"><div class="li"> Add different concentrations of CoCl2 (50µM, 100µM and 200 µM)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Observe the cells under fluorescence microscope 3h, 6h, 8h, 16h, 18h and 24h after CoCl2 treatment</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> No detectable induction of gene expression for every approach</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_105'></div>
+
−
<h2 class="sectionedit106" id="section040717">04.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_106'></div>
+
−
<h3 class="sectionedit107" id="lentiviral_transduction_plenti-crispr_from_ag_cathomen">Lentiviral transduction (pLenti-CRISPR from AG Cathomen)</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Change the medium and add puromycin for selection</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_107'></div>
+
−
<h3 class="sectionedit108" id="biorad_electroporationrepeat_pgfp_transfection1">BioRad Electroporation: Repeat pGFP transfection</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
<strong>9µg for each approach</strong><br/>
+
−
 
+
−
</p>
+
−
<ul>
+
−
<li class="level1"><div class="li"> Setting: 12 well plate, 1 well set, 1 pulse</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit109"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Approach</th><th class="col1">Capacitance[µF]</th><th class="col2">Voltage</th><th class="col3">Resistance</th><th class="col4">Time Constance [msec]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1: no plasmid</td><td class="col1">950</td><td class="col2">250</td><td class="col3">∞</td><td class="col4">20</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2: Mock plasmid</td><td class="col1">950</td><td class="col2">250</td><td class="col3">∞</td><td class="col4">20</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">3: pIG17_009</td><td class="col1">950</td><td class="col2">250</td><td class="col3">∞</td><td class="col4">20</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">4: pIG17_009</td><td class="col1">950</td><td class="col2">250</td><td class="col3">500Ω</td><td class="col4">20</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">5: pIG17_082 (p526)</td><td class="col1">950</td><td class="col2">250</td><td class="col3">∞</td><td class="col4">20</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_109'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_108'></div>
+
−
<h2 class="sectionedit110" id="section050717">05.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_110'></div>
+
−
<h3 class="sectionedit111" id="pei_transfection_pig17_022_and_pig17_023_for_ctla-4_promoter_test">PEI transfection (pIG17_022 and pIG17_023) for CTLA-4 promoter test</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> In total: 3*6 wells </div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfection with pIG17_009 as positive control: 1 well</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfection with pIG17_022: 4 wellS</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfection with pIG17_023: 4 wellS</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Per Approach: 3µg DNA, 200µl serum free DMEM and 9µg PEI</div>
+
−
</li>
+
−
</ul>
+
−
<ol>
+
−
<li class="level1"><div class="li"> Split the cells 1:5 into 10 cm plate (on 03.07.17)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Thaw PEI reagent at RT</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Dilute plasmid in 200µl serum free DMEM (3µg from each plasmid)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add PEI(1µg/µl) to the mix while vortexing (PEI:DNA=3:1)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> incubate 15 min at RT</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add 200µl mix to wells</div>
+
−
</li>
+
−
</ol>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_111'></div>
+
−
<h3 class="sectionedit112" id="flow_cytometry1">Flow Cytometry</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Cells transfected with pIG17_082 showed high transfection efficiency</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Cells transfected with our stock pIG17_009 showed low efficiency and low survival rate</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_112'></div>
+
−
<h2 class="sectionedit113" id="section060717">06.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_113'></div>
+
−
<h3 class="sectionedit114" id="prepare_cells_for_lentivirus-production">Prepare cells for lentivirus-production</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Plate about 4 mio. cells, make replicate</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_114'></div>
+
−
<h3 class="sectionedit115" id="vegf_induction_with_modeling_group">VEGF induction with Modeling group</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> No significant induction</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_115'></div>
+
−
<h2 class="sectionedit116" id="section070717">07.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_116'></div>
+
−
<h3 class="sectionedit117" id="lentivirus_pig17_119_production_in_hek_cells">Lentivirus(pIG17_119) production in HEK cells</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
Transfection of HEK cells for virus production.
+
−
</p>
+
−
<div class="table sectionedit118"><table class="inline">
+
−
<tr class="row0">
+
−
<td class="col0"><strong>Lentiviral packaging cells</strong></td><td class="col1">Hek293T cells</td>
+
−
</tr>
+
−
<tr class="row1">
+
−
<td class="col0"><strong>Plate size</strong></td><td class="col1">10 cm</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0"><strong>Total DNA/plate</strong></td><td class="col1">12.87 µg</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0"><strong>Transfer: gag/pol: env ratio</strong></td><td class="col1">2:1:1</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0"><strong>Construct</strong></td><td class="col1">6.43 µg</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0"><strong>pCMV∆R8.74</strong></td><td class="col1">3.22 µg</td>
+
−
</tr>
+
−
<tr class="row6">
+
−
<td class="col0"><strong>pMD2G</strong></td><td class="col1">3.22 µg</td>
+
−
</tr>
+
−
<tr class="row7">
+
−
<td class="col0"><strong>N/P ration</strong></td><td class="col1">15</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_118'></div><div class="table sectionedit119"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Plasmid number</th><th class="col1">component</th><th class="col2">concentration[ng/µL]</th><th class="col3">volume [µL]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">pIG17_119</td><td class="col1"><strong>pCRE_GFP_CMV_mCherry</strong></td><td class="col2">167</td><td class="col3">38.5</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">pIG17_003</td><td class="col1"><strong>Envelope plasmid pMD2G</strong></td><td class="col2">243.4</td><td class="col3">13.3</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">pIG17_004</td><td class="col1"><strong>Packaging Plasmid pCMV∆R8.74</strong></td><td class="col2">819</td><td class="col3">4</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_119'></div><ul>
+
−
<li class="level1"><div class="li"> Packaging Mastermix</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit120"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Component</th><th class="col1">1x [µL]</th><th class="col2">2.1x [µL]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">pIG17_003</td><td class="col1">13.3</td><td class="col2">28</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">pIG17_004</td><td class="col1">4</td><td class="col2">8.4</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">150mM NaCl</td><td class="col1">441.49</td><td class="col2">927.12</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">total</td><td class="col1">458.8</td><td class="col2">963.52</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_120'></div><ul>
+
−
<li class="level1"><div class="li"> PEI Mastermix</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit121"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Component</th><th class="col1">1x [µL]</th><th class="col2">2.1x [µL]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">23.2mM (1µg/µl)PEI</td><td class="col1">24.94</td><td class="col2">52.41</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">150mM NaCl</td><td class="col1">475.04</td><td class="col2">997.59</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">total</td><td class="col1">499.98</td><td class="col2">1050</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_121'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_117'></div>
+
−
<h2 class="sectionedit122" id="section080717">08.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_122'></div>
+
−
<h3 class="sectionedit123" id="lentivirus_pig17_119_production_in_hek_cells1">Lentivirus(pIG17_119) production in HEK cells</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Change the medium</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_123'></div>
+
−
<h3 class="sectionedit124" id="biorad_electroporation9">BioRad electroporation</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
<strong>4 Approaches (9µg/Approach)</strong>
+
−
<strong>Setting: 12 well plate, 1 well set, 1 Pulse, ∞ Resistance</strong>
+
−
</p>
+
−
<ol>
+
−
<li class="level1"><div class="li"> Electroporated without plasmid</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> CMV-GFP from Nicole (700 ng/µl)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> CMV-GFP (Yael, 06.06.17, 140.4 ng/µl)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> CMV-GFP (Dennis, 27.06.17, Dennis, 86.8 ng/µl)</div>
+
−
</li>
+
−
</ol>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_124'></div>
+
−
<h2 class="sectionedit125" id="section090717">09.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_125'></div>
+
−
<h3 class="sectionedit126" id="lentivirus_pig17_119_production_in_hek_cells2">Lentivirus(pIG17_119) production in HEK cells</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Collect virus medium</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add new medium</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_126'></div>
+
−
<h2 class="sectionedit127" id="section100717">10.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_127'></div>
+
−
<h3 class="sectionedit128" id="lentivirus_pig17_119_production_in_hek_cells3">Lentivirus(pIG17_119) production in HEK cells</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Collect virus medium</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add new medium</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_128'></div>
+
−
<h2 class="sectionedit129" id="section110717">11.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_129'></div>
+
−
<h3 class="sectionedit130" id="pei_transfection_ctla-4_test">PEI transfection (CTLA-4 Test)</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> In total: 3*6 wells </div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfection with pIG17_009 as positive control: 1 well (plasmid from Nicole); 1 well (Culture from Nicole, from us preped using promega miniprep)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfection with pIG17_022: 5 wellS</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfection with pIG17_023: 5 wellS</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Per Approach: 3µg DNA, 200µl serum free DMEM and 9µg PEI</div>
+
−
</li>
+
−
</ul>
+
−
<ol>
+
−
<li class="level1"><div class="li"> Split the cells 1:5 into 10 cm plate (on 03.07.17)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Thaw PEI reagent at RT</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Dilute plasmid in 200µl serum free DMEM (3µg from each plasmid)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add PEI(1µg/µl) to the mix while vortexing (PEI:DNA=3:1)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> incubate 15 min at RT</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add 200µl mix to wells</div>
+
−
</li>
+
−
</ol>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_130'></div>
+
−
<h3 class="sectionedit131" id="lentiviral_transduction_pig17_119_of_jurkat_cells">Lentiviral transduction (pIG17_119) of Jurkat cells</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Use 6-well plate</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> plate 200000 cells/well</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Make Titer test with virus medium </div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
<p>
+
−
<strong>The cells were not successfully infected.</strong>
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_131'></div>
+
−
<h2 class="sectionedit132" id="section120717">12.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_132'></div>
+
−
<h3 class="sectionedit133" id="biorad_electroporation10">BioRad electroporation</h3>
+
−
<div class="level3">
+
−
<div class="table sectionedit134"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Approach</th><th class="col1">plasmid</th><th class="col2">Capacitance[µF]</th><th class="col3">Voltage</th><th class="col4">Resistance</th><th class="col5">Time Constance [msec]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1</td><td class="col1"> no plasmid</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2</td><td class="col1"> CMV-GFP (Nicole)</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">3</td><td class="col1"> CMV-GFP (Culture from Nicole, promega mini)</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">4</td><td class="col1"> pIG17_104: Cas9-GFP-gRNA3</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_134'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_133'></div>
+
−
<h2 class="sectionedit135" id="section140717">14.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_135'></div>
+
−
<h3 class="sectionedit136" id="lentivirus_pig17_121_production_in_hek_cells">Lentivirus(pIG17_121) production in HEK cells</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
Transfection of HEK cells for virus production.
+
−
</p>
+
−
<div class="table sectionedit137"><table class="inline">
+
−
<tr class="row0">
+
−
<td class="col0"><strong>Lentiviral packaging cells</strong></td><td class="col1">Hek293T cells</td>
+
−
</tr>
+
−
<tr class="row1">
+
−
<td class="col0"><strong>Plate size</strong></td><td class="col1">10 cm</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0"><strong>Total DNA/plate</strong></td><td class="col1">12.87 µg</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0"><strong>Transfer: gag/pol: env ratio</strong></td><td class="col1">2:1:1</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0"><strong>Construct</strong></td><td class="col1">6.43 µg</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0"><strong>pCMV∆R8.74</strong></td><td class="col1">3.22 µg</td>
+
−
</tr>
+
−
<tr class="row6">
+
−
<td class="col0"><strong>pMD2G</strong></td><td class="col1">3.22 µg</td>
+
−
</tr>
+
−
<tr class="row7">
+
−
<td class="col0"><strong>N/P ration</strong></td><td class="col1">15</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_137'></div><div class="table sectionedit138"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Plasmid number</th><th class="col1">component</th><th class="col2">concentration[ng/µL]</th><th class="col3">volume [µL]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">pIG17_121</td><td class="col1"><strong>pCTLA4(380)_GFP_CMV_mCherry</strong></td><td class="col2">1682</td><td class="col3">3.82</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">pIG17_003</td><td class="col1"><strong>Envelope plasmid pMD2G</strong></td><td class="col2">321.9</td><td class="col3">10</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">pIG17_004</td><td class="col1"><strong>Packaging Plasmid pCMV∆R8.74</strong></td><td class="col2">819</td><td class="col3">4</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_138'></div><ul>
+
−
<li class="level1"><div class="li"> Packaging Mastermix</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit139"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Component</th><th class="col1">1x [µL]</th><th class="col2">2.1x [µL]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">pIG17_003</td><td class="col1">10</td><td class="col2">21</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">pIG17_004</td><td class="col1">4</td><td class="col2">8.4</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">150mM NaCl</td><td class="col1">441.49</td><td class="col2">927.12</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">pIG17_121</td><td class="col1">3.82</td><td class="col2">8</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">total</td><td class="col1">504.95</td><td class="col2">1060</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_139'></div><ul>
+
−
<li class="level1"><div class="li"> PEI Mastermix</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit140"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Component</th><th class="col1">1x [µL]</th><th class="col2">2.1x [µL]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">23.2mM (1µg/µl)PEI</td><td class="col1">24.94</td><td class="col2">52.41</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">150mM NaCl</td><td class="col1">475.04</td><td class="col2">997.59</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">total</td><td class="col1">499.98</td><td class="col2">1050</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_140'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_136'></div>
+
−
<h3 class="sectionedit141" id="biorad_electroporation11">BioRad electroporation</h3>
+
−
<div class="level3">
+
−
<div class="table sectionedit142"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Approach</th><th class="col1">plasmid</th><th class="col2">Capacitance[µF]</th><th class="col3">Voltage</th><th class="col4">Resistance</th><th class="col5">Time Constance [msec]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1</td><td class="col1"> no plasmid</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2</td><td class="col1"> 6µg Cas9-GFP-gRNA3</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">3</td><td class="col1"> 9µg Cas9-GFP-gRNA3</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">4</td><td class="col1"> 12µg Cas9-GFP-gRNA3</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_142'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_141'></div>
+
−
<h2 class="sectionedit143" id="section150717">15.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_143'></div>
+
−
<h3 class="sectionedit144" id="collect_virus_pig17_121">collect virus (pIG17_121)</h3>
+
−
<div class="level3">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_144'></div>
+
−
<h3 class="sectionedit145" id="flow_cytometry_biorad_7_14">flow cytometry (BioRad 7/14)</h3>
+
−
<div class="level3">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_145'></div>
+
−
<h2 class="sectionedit146" id="section160717">16.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_146'></div>
+
−
<h3 class="sectionedit147" id="pei_transfection">PEI transfection</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> In total: 3*6 wells </div>
+
−
</li>
+
−
<li class="level1"><div class="li"> One well with non-transfected cells </div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfection with CMV-GFP as positive control: 1 well (JetStar); 1 well (Promega Mini)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfection with pIG17_013: 4 wellS</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Per Approach: 3µg DNA, 200µl serum free DMEM and 9µg PEI</div>
+
−
</li>
+
−
</ul>
+
−
<ol>
+
−
<li class="level1"><div class="li"> Split the cells 1:5 into 6-well plate (on 15.07.17)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Thaw PEI reagent at RT</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Dilute plasmid in 200µl serum free DMEM (3µg from each plasmid)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add PEI(1µg/µl) to the mix while vortexing (PEI:DNA=3:1)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> incubate 15 min at RT</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add 200µl mix to wells</div>
+
−
</li>
+
−
</ol>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_147'></div>
+
−
<h3 class="sectionedit148" id="biorad_electroporation12">BioRad electroporation</h3>
+
−
<div class="level3">
+
−
<div class="table sectionedit149"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Approach</th><th class="col1">plasmid</th><th class="col2">Capacitance[µF]</th><th class="col3">Voltage</th><th class="col4">Resistance</th><th class="col5">Time Constance [msec]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1</td><td class="col1"> no plasmid</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2</td><td class="col1"> CMV-GFP JetStar</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">3</td><td class="col1"> CMV-GFP promega mini</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">4</td><td class="col1"> CMV-mCherry (Nicole)</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">5</td><td class="col1"> pIG17_008 (Jana)</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_149'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_148'></div>
+
−
<h2 class="sectionedit150" id="section170717">17.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_150'></div>
+
−
<h3 class="sectionedit151" id="hypoxia_test_in_hypoxia_incubator_ag_cathomen">Hypoxia Test in hypoxia incubator (AG Cathomen)</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> with 1%O2</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_151'></div>
+
−
<h3 class="sectionedit152" id="flow_cytometry2">Flow Cytometry</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> BioRad didn&#039;t work</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_152'></div>
+
−
<h2 class="sectionedit153" id="section180717">18.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_153'></div>
+
−
<h3 class="sectionedit154" id="hypoxia_test_in_hypoxia_incubator_ag_cathomen1">Hypoxia Test in hypoxia incubator (AG Cathomen)</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Fluorescence microscopy</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> After 24h incubation: cells showed normal morphology and there was no autofluorescence.</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_154'></div>
+
−
<h3 class="sectionedit155" id="lentiviral_transduction3">Lentiviral transduction</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> wash the virus-transducted cells (pIG17_119)</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_155'></div>
+
−
<h3 class="sectionedit156" id="biorad_electroporation13">BioRad electroporation</h3>
+
−
<div class="level3">
+
−
<div class="table sectionedit157"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Approach</th><th class="col1">plasmid</th><th class="col2">Capacitance[µF]</th><th class="col3">Voltage</th><th class="col4">Resistance</th><th class="col5">Time Constance [msec]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1</td><td class="col1"> pIG17_119</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2</td><td class="col1"> pIG17_121</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">3</td><td class="col1"> pIG17_031</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">4</td><td class="col1"> pIG17_034</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">5</td><td class="col1"> pIG17_037</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row6">
+
−
<td class="col0">5</td><td class="col1"> pIG17_086</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_157'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_156'></div>
+
−
<h3 class="sectionedit158" id="pei_transfection_ctla-4_test1">PEI transfection (CTLA-4 Test)</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> In total: 2*12 wells </div>
+
−
</li>
+
−
<li class="level1"><div class="li"> One well with non-transfected cells </div>
+
−
</li>
+
−
<li class="level1"><div class="li"> One well: Transfection with CMV-GFP as positive control</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfection with pIG17_022: 5 wellS</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfection with pIG17_023: 5 wellS</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfection with pIG17_037: 5 wellS</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfection with pIG17_086: 5 wellS</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Per Approach: 1.5µg DNA, 100µl serum free DMEM and 4.5µg PEI</div>
+
−
</li>
+
−
</ul>
+
−
<ol>
+
−
<li class="level1"><div class="li"> Split the cells 1:10 into 12-well plate (on 17.07.17)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Thaw PEI reagent at RT</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Dilute plasmid in 100µl serum free DMEM (3µg from each plasmid)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add PEI(1µg/µl) to the mix while vortexing (PEI:DNA=3:1)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> incubate 15 min at RT</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add 100 µl mix to wells</div>
+
−
</li>
+
−
</ol>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_158'></div>
+
−
<h3 class="sectionedit159" id="pei_transfection_hypoxia_test_with_cocl2">PEI transfection (hypoxia test with CoCl2)</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> In total: 4*6-well plates </div>
+
−
</li>
+
−
<li class="level1"><div class="li"> One well in each plate: with non-transfected cells </div>
+
−
</li>
+
−
<li class="level1"><div class="li"> One well in each plate: Transfection with CMV-GFP as positive control</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfection with pIG17_013: 8 wellS</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Transfection with pIG17_031: 8 wellS</div>
+
−
</li>
+
−
</ul>
+
−
<ol>
+
−
<li class="level1"><div class="li"> Split the cells 1:10 into 6-well plate (on 17.07.17)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Thaw PEI reagent at RT</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Dilute plasmid in 100µl serum free DMEM (3µg from each plasmid)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add PEI(1µg/µl) to the mix while vortexing (PEI:DNA=3:1)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> incubate 15 min at RT</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add 200 µl mix to wells</div>
+
−
</li>
+
−
</ol>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_159'></div>
+
−
<h2 class="sectionedit160" id="section190717">19.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_160'></div>
+
−
<h3 class="sectionedit161" id="concentrate_the_viruses_pig17_119_and_pig17_121">Concentrate the viruses (pIG17_119 and pIG17_121)</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> for 20 ml virus medium, use 5ml 20% sucrose</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> carefully add virus medium to sucrose (very tricky)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> centrifuge at 4000 rpm over night</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> After centrifugation: remove the medium, put the falcon tube upside down and air dry.</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> slowly add 100 µl PBS (20-30x, also very tricky) </div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Incubate the pellet for 30-60 min</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> resuspend the cells 2x </div>
+
−
</li>
+
−
<li class="level1"><div class="li"> aliquot the virus</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> store in -80°C freezer</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_161'></div>
+
−
<h3 class="sectionedit162" id="lentiviral_transduction4">Lentiviral transduction</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> plate 200000 Jurkat cells/ well in one 6-well plate</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> add the concentrated virus (pIG17_119 and pIG17_121) (30µl and 50µl)</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_162'></div>
+
−
<h3 class="sectionedit163" id="cocl2_treatment_24h_after_pei">CoCl2 treatment (24h after PEI)</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Treatment with 2*6 well plate: 1 with pIG17_013 and 1 with pIG17_031</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Make new 2M CoCl2 stock</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> add CoCl2 to the cells, end-concentration: 0, 300µM, 500µM and 100µM</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Incubation for 24h and track the induction of cell expression</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_163'></div>
+
−
<h3 class="sectionedit164" id="hypoxia_test_in_hypoxia_incubator_ag_cathomen2">Hypoxia Test in hypoxia incubator (AG Cathomen)</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Fluorescence microscopy</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> After 48h incubation: cells were mostly dead showing autofluorescence.</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_164'></div>
+
−
<h3 class="sectionedit165" id="flow_cytometry_biorad_on_july_20th">Flow Cytometry (BioRad on July 20th)</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> For mCherry excitation: FL3!!! </div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_165'></div>
+
−
<h2 class="sectionedit166" id="section200717">20.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_166'></div>
+
−
<h3 class="sectionedit167" id="cocl2_treatment_48h_after_pei">CoCl2 treatment (48h after PEI)</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Treatment with 2*6 well plate: 1 with pIG17_013 and 1 with pIG17_031</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Make new 2M CoCl2 stock</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> add CoCl2 to the cells, end-concentration: 0, 300µM, 500µM and 100µM</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Incubation for 24h and track the induction of cell expression</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_167'></div>
+
−
<h3 class="sectionedit168" id="hypoxia_test_in_hypoxia_incubator_ag_cathomen3">Hypoxia Test in hypoxia incubator (AG Cathomen)</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Fluorescence microscopy</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> After 72h incubation: cells were mostly dead showing autofluorescence.</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_168'></div>
+
−
<h2 class="sectionedit169" id="section210717">21.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_169'></div>
+
−
<h3 class="sectionedit170" id="biorad_electroporation_knock-out_plasmids">BioRad Electroporation (Knock-Out plasmids)</h3>
+
−
<div class="level3">
+
−
<div class="table sectionedit171"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Approach</th><th class="col1">plasmid</th><th class="col2">Capacitance[µF]</th><th class="col3">Voltage</th><th class="col4">Resistance</th><th class="col5">Time Constance [msec]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1</td><td class="col1"> -</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2</td><td class="col1"> CMV-GFP (#503 from Nicole)</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">3</td><td class="col1"> pIG17_082 (p526)</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">4</td><td class="col1"> Cas9-GFP-gRNA5</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">5</td><td class="col1"> Cas9-GFP-scramble</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_171'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_170'></div>
+
−
<h2 class="sectionedit172" id="section220717">22.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_172'></div>
+
−
<h3 class="sectionedit173" id="lentivirus_pig17_119_and_pig17_121_production_in_hek_cells">Lentivirus(pIG17_119 and pIG17_121) production in HEK cells</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
<strong>PEI Transfection of HEK cells for virus production.</strong>
+
−
</p>
+
−
<div class="table sectionedit174"><table class="inline">
+
−
<tr class="row0">
+
−
<td class="col0"><strong>Lentiviral packaging cells</strong></td><td class="col1">Hek293T cells</td>
+
−
</tr>
+
−
<tr class="row1">
+
−
<td class="col0"><strong>Plate size</strong></td><td class="col1">10 cm</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0"><strong>Total DNA/plate</strong></td><td class="col1">12.87 µg</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0"><strong>Transfer: gag/pol: env ratio</strong></td><td class="col1">2:1:1</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0"><strong>Construct</strong></td><td class="col1">6.43 µg</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0"><strong>pCMV∆R8.74</strong></td><td class="col1">3.22 µg</td>
+
−
</tr>
+
−
<tr class="row6">
+
−
<td class="col0"><strong>pMD2G</strong></td><td class="col1">3.22 µg</td>
+
−
</tr>
+
−
<tr class="row7">
+
−
<td class="col0"><strong>N/P ration</strong></td><td class="col1">15</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_174'></div><div class="table sectionedit175"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Plasmid number</th><th class="col1">component</th><th class="col2">concentration[ng/µL]</th><th class="col3">volume [µL]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">pIG17_119</td><td class="col1"><strong>pCRE_GFP_CMV_mCherry</strong></td><td class="col2">167</td><td class="col3">38</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">pIG17_121</td><td class="col1"><strong>pCTLA4(380)_GFP_CMV_mCherry</strong></td><td class="col2">1682</td><td class="col3">3.82</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">pIG17_003</td><td class="col1"><strong>Envelope plasmid pMD2G</strong></td><td class="col2">105</td><td class="col3">30.7</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">pIG17_004</td><td class="col1"><strong>Packaging Plasmid pCMV∆R8.74</strong></td><td class="col2">208</td><td class="col3">15.5</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_175'></div><ul>
+
−
<li class="level1"><div class="li"> Packaging Mastermix</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit176"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Component</th><th class="col1">1x [µL]</th><th class="col2">2.1x [µL]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">pIG17_003</td><td class="col1">30.7</td><td class="col2">64.5</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">pIG17_004</td><td class="col1">15.5</td><td class="col2">32.6</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">150mM NaCl</td><td class="col1">441.49</td><td class="col2">927.12</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_176'></div><ul>
+
−
<li class="level1"><div class="li"> PEI Mastermix</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit177"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Component</th><th class="col1">1x [µL]</th><th class="col2">2.1x [µL]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">23.2mM (1µg/µl)PEI</td><td class="col1">24.94</td><td class="col2">52.41</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">150mM NaCl</td><td class="col1">475.04</td><td class="col2">997.59</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">total</td><td class="col1">499.98</td><td class="col2">1050</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_177'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_173'></div>
+
−
<h2 class="sectionedit178" id="section2207171">22.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_178'></div>
+
−
<h3 class="sectionedit179" id="flow_cytometrybiorad_electroporation_7_21">Flow Cytometry: BioRad Electroporation 7/21</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
<strong>there was GFP positive signals during flow cytometry measurement but the cells were mycoplasma positive</strong>
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_179'></div>
+
−
<h3 class="sectionedit180" id="lentivirus_pig17_119_and_pig17_121_production_in_hek_cells1">Lentivirus(pIG17_119 and pIG17_121) production in HEK cells</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Collect virus</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_180'></div>
+
−
<h2 class="sectionedit181" id="section230717">23.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_181'></div>
+
−
<h3 class="sectionedit182" id="lentivirus_pig17_119_and_pig17_121_production_in_hek_cells2">Lentivirus(pIG17_119 and pIG17_121) production in HEK cells</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Collect virus</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_182'></div>
+
−
<h2 class="sectionedit183" id="section240717">24.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_183'></div>
+
−
<h3 class="sectionedit184" id="mycoplamsa_test">Mycoplamsa Test</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Both Cas9-GFP-gRNA5 and Cas9-GFP-scramble transfected cells were mycoplasma positive</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_184'></div>
+
−
<h2 class="sectionedit185" id="section250717">25.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_185'></div>
+
−
<h3 class="sectionedit186" id="pei_transfection_of_cho_cellshypoxia_tet_system">PEI transfection of CHO cells: Hypoxia &amp; Tet system</h3>
+
−
<div class="level3">
+
−
<ol>
+
−
<li class="level1"><div class="li"> Split the cells 1:2 in 12-well plate (on 24.07.17)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Per Approach: 1µg DNA + 8µg PEI and fill up to 100 µl with serum free DMEM</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Thaw PEI reagent at RT</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add PEI(1µg/µl) to the mix while vortexing </div>
+
−
</li>
+
−
<li class="level1"><div class="li"> incubate 15 min at RT</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add the mix to cells</div>
+
−
</li>
+
−
</ol>
+
−
<div class="table sectionedit187"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Plasmid</th><th class="col1">Approach</th><th class="col2">DNA Concentration[ng/µl]</th><th class="col3">DNA volume</th><th class="col4">Volume of DMEM[µl]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">CMV-GFP (#503 from Nicole)</td><td class="col1"> 1 </td><td class="col2">25.5</td><td class="col3">40</td><td class="col4">52</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">CMV-mCherry (AG Hiltbrunner)</td><td class="col1"> 1</td><td class="col2">1200</td><td class="col3">1</td><td class="col4">91</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">pIG17_012</td><td class="col1">6</td><td class="col2">253</td><td class="col3">24</td><td class="col4">528</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">pIG17_013</td><td class="col1">6</td><td class="col2">583.3</td><td class="col3">10.3</td><td class="col4">541.7</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">pIG17_031</td><td class="col1">6</td><td class="col2">746.9</td><td class="col3">8</td><td class="col4">544</td>
+
−
</tr>
+
−
<tr class="row6">
+
−
<td class="col0">Tet-GFP (Shima)</td><td class="col1">2 (protocol from Shima)</td><td class="col2">520</td><td class="col3">3.8</td><td class="col4">180.2</td>
+
−
</tr>
+
−
<tr class="row7">
+
−
<td class="col0">Tet-GFP (Shima)</td><td class="col1">2 (our protocol)</td><td class="col2">520</td><td class="col3">5.8</td><td class="col4">200</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_187'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_186'></div>
+
−
<h3 class="sectionedit188" id="tetracycline_induction_of_cho">Tetracycline induction of CHO</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> 3h after PEI transfection: remove media, add tetracycline to new media (1:1000 dilution)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Tetracycline Stock: 2mg/ml (End: 1µg per well of 12-well plate)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 3h after adding tetracycline: no induction</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> incubation till the next day: microscopic observation</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Gene expression was induced by tetracycline compared to the tetracycline-untreated cells</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_188'></div>
+
−
<h3 class="sectionedit189" id="pei_transfection_of_hek_cellshypoxia">PEI transfection of HEK cells: Hypoxia</h3>
+
−
<div class="level3">
+
−
<ol>
+
−
<li class="level1"><div class="li"> Split the cells 1:5 in 12-well plate (on 24.07.17)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Per Approach: 1.5µg DNA + 4.5µg PEI and 100 µl serum free DMEM</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Thaw PEI reagent at RT</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add PEI(1µg/µl) to the mix while vortexing </div>
+
−
</li>
+
−
<li class="level1"><div class="li"> incubate 15 min at RT</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add the mix to cells</div>
+
−
</li>
+
−
</ol>
+
−
<div class="table sectionedit190"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Plasmid</th><th class="col1">Approach</th><th class="col2">DNA Concentration[ng/µl]</th><th class="col3">DNA volume</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">CMV-GFP (#503 from Nicole)</td><td class="col1"> 1 </td><td class="col2">25.5</td><td class="col3">59</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">CMV-mCherry (AG Hiltbrunner)</td><td class="col1"> 1</td><td class="col2">1200</td><td class="col3">1.25</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">pIG17_012</td><td class="col1">6</td><td class="col2">131.4</td><td class="col3">12</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">pIG17_013</td><td class="col1">6</td><td class="col2">583.3</td><td class="col3">15.5</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">pIG17_031</td><td class="col1">6</td><td class="col2">746.9</td><td class="col3">12</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_190'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_189'></div>
+
−
<h2 class="sectionedit191" id="section260717">26.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_191'></div>
+
−
<h3 class="sectionedit192" id="cocl2_treatment">CoCl2 treatment</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Add corresponding concentration of CoCl2 to (hypoxic plasmid) transfected cells (CHO and HEK)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> CoCl2 stock: 2M </div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit193"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Endconcentration of CoCl2[µM]</th><th class="col1">Volume from Stock[µl]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">0</td><td class="col1">0</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">100</td><td class="col1">0.1</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">200</td><td class="col1">0.2</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">300</td><td class="col1">0.3</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">500</td><td class="col1">0.5</td>
+
−
</tr>
+
−
<tr class="row6">
+
−
<td class="col0">1000</td><td class="col1">1</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_193'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_192'></div>
+
−
<h2 class="sectionedit194" id="section270717">27.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_194'></div>
+
−
<h3 class="sectionedit195" id="seap_assay_with_hek_cells">SEAP assay with HEK cells</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
- gather 200 µl of cell supernatant <br/>
+
−
 
+
−
- incubate cell supernatant at 65°C for 30min <br/>
+
−
 
+
−
- centrifuge cell supernatant for 1 min at 1250g <br/>
+
−
 
+
−
- add 150 µl supernatant into a cuvette <br/>
+
−
 
+
−
- add 500 µl 2x SEAP buffer <br/>
+
−
 
+
−
- add 100 µl pNPP and remove bubbles carefully <br/>
+
−
 
+
−
- measure in a nano drop every 5min for 2h <br/>
+
−
 
+
−
<img src="https://static.igem.org/mediawiki/2017/8/8c/7_27_2017_SEAP_assay.png">
+
−
 
+
−
<a href="/igem2017/lib/exe/detail.php?id=labor%3Acell_culture&amp;media=labor:screen_shot_2017-08-22_at_13.57.57.png" class="media" title="labor:screen_shot_2017-08-22_at_13.57.57.png"><img src="/igem2017/lib/exe/fetch.php?media=labor:screen_shot_2017-08-22_at_13.57.57.png" class="media" alt="" /></a>
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_195'></div>
+
−
<h3 class="sectionedit196" id="replace_stock_cultures_with_new_culture_from_toolbox">Replace Stock cultures with new culture from Toolbox</h3>
+
−
<div class="level3">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_196'></div>
+
−
<h2 class="sectionedit197" id="section310717">31.07.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_197'></div>
+
−
<h3 class="sectionedit198" id="mycoplasma_testnew_hek_stock">Mycoplasma Test: New HEK stock</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> New HEK cells were mycoplasma negative</div>
+
−
</li>
+
−
</ul>
+
−
            </div>           
+
−
            <br>
+
−
        </div>
+
−
               
+
−
             
+
−
<button class="accordion">August</button>
+
−
        <div class="panel panel-default">
+
−
            <br>
+
−
 
+
−
           
+
−
<div class='secedit editbutton_section editbutton_198'></div>
+
−
<h2 class="sectionedit199" id="section010817">01.08.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_199'></div>
+
−
<h3 class="sectionedit200" id="biorad_electroporation14">BioRad Electroporation</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Per Approach: 9µg DNA, 2 mio.JK cells</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit201"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Approach</th><th class="col1">plasmid</th><th class="col2">Capacitance[µF]</th><th class="col3">Voltage</th><th class="col4">Resistance</th><th class="col5">Time Constance [msec]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1</td><td class="col1">pIG17_009 (06.06 Yael)</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2</td><td class="col1">Cas9-GFP-gRNA5</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">3</td><td class="col1">Cas9-GFP-scramble</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">4</td><td class="col1">KO-Kit: 203+101</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">5</td><td class="col1">KO-Kit: 203+104</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row6">
+
−
<td class="col0">6</td><td class="col1">KO-Kit scramble</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_201'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_200'></div>
+
−
<h3 class="sectionedit202" id="pei_transfectionhypoxia_-_seap_assay_in_hek_cells">PEI transfection: Hypoxia - SEAP assay in HEK cells</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Per Approach: 1.5µg DNA, 4.5µg PEI, 100µl DMEM</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit203"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Plasmid number</th><th class="col1">component</th><th class="col2">concentrations of the used tubes[ng/µL]</th><th class="col3">volumes from the used tubes[µL] for 36µg</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">pIG17_013</td><td class="col1"><strong>HRE-SEAP</strong></td><td class="col2">583.3 and 88.1</td><td class="col3">55.7 and 40</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">pIG17_002</td><td class="col1"><strong>CMV-SEAP</strong></td><td class="col2">92.0 and 91.4</td><td class="col3">350 and 41.6</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_203'></div>
+
−
<p>
+
−
Due to the concentrations and volumes in the tubes for each approach two were used.
+
−
Per construct 24 wells were used.
+
−
Each well will have different CoCl2 concentrations. For each approach triplicates will be analysed.
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_202'></div>
+
−
<h2 class="sectionedit204" id="section020817">02.08.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_204'></div>
+
−
<h3 class="sectionedit205" id="flow_cytometry_of_biorad_electroporation_8_1ko_plasmids">Flow Cytometry of BioRad Electroporation (8/1): KO plasmids</h3>
+
−
<div class="level3">
+
−
<img src="https://static.igem.org/mediawiki/2017/8/85/8_2_2017_Flow_Cytometry.jpeg">
+
−
 
+
−
<p>
+
−
<a href="/igem2017/lib/exe/detail.php?id=labor%3Acell_culture&amp;media=labor:flowjo_ko_02-aug-2017-layout.jpg" class="media" title="labor:flowjo_ko_02-aug-2017-layout.jpg"><img src="/igem2017/lib/exe/fetch.php?w=1100&amp;tok=8c31e0&amp;media=labor:flowjo_ko_02-aug-2017-layout.jpg" class="media" alt="" width="1100" /></a>
+
−
</p>
+
−
 
+
−
<p>
+
−
For the knockout Kit we could see the most living GFP positive cells (26.2%) for the cotransfection of 203 and 104.
+
−
The Cas9 GFP guide RNA5 had 5.21% GFP positive cells under the gated living cells.
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_205'></div>
+
−
<h3 class="sectionedit206" id="addition_of_cocl2hypoxia_-_seap_assay_in_hek_cells">Addition of CoCl2: Hypoxia - SEAP assay in HEK cells</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
1:10 dilution of a 2M CoCl2 stock with PBS. CoCl2 was added to HRE-SEAP approaches in order to induce the promoter, as well as to CMV-SEAP approaches
+
−
</p>
+
−
 
+
−
<p>
+
−
Approaches:
+
−
For each approach triplicates were done.
+
−
</p>
+
−
<div class="table sectionedit207"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">CoCL2 concentration[µM]</th><th class="col1">volume[µL] of 200mM CoCl2 </th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">0</td><td class="col1">0</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">100</td><td class="col1">0.55</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">150</td><td class="col1">0.825</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">200</td><td class="col1">1.1</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">250</td><td class="col1">1.375</td>
+
−
</tr>
+
−
<tr class="row6">
+
−
<td class="col0">300</td><td class="col1">1.65</td>
+
−
</tr>
+
−
<tr class="row7">
+
−
<td class="col0">500</td><td class="col1">2.75</td>
+
−
</tr>
+
−
<tr class="row8">
+
−
<td class="col0">1000</td><td class="col1">5.5</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_207'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_206'></div>
+
−
<h3 class="sectionedit208" id="lentivirus_production_in_hek_cellsmock_knock-down_plasmid_114_115_116_pig17_119_pig17_121">Lentivirus production in HEK cells: Mock, Knock-down plasmid (114,115,116), pIG17_119, pIG17_121</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
<strong>Follow the protocol from Frederike (AG Schamel)</strong>
+
−
<strong>PEI Transfection of HEK cells for virus production.</strong>
+
−
</p>
+
−
<ol>
+
−
<li class="level1"><div class="li"> Prepare packaging and PEI mix separately and incubate for 10 min</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Mix packaging mix with transferplasmid</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add PEI mix to DNA mix and incubate for 15 min</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add the PEI+DNA mix to cells</div>
+
−
</li>
+
−
</ol>
+
−
<div class="table sectionedit209"><table class="inline">
+
−
<tr class="row0">
+
−
<td class="col0"><strong>Lentiviral packaging cells</strong></td><td class="col1">Hek293T cells</td>
+
−
</tr>
+
−
<tr class="row1">
+
−
<td class="col0"><strong>Plate size</strong></td><td class="col1">10 cm</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0"><strong>Total DNA/plate</strong></td><td class="col1">12.87 µg</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0"><strong>Transfer: gag/pol: env ratio</strong></td><td class="col1">2:1:1</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0"><strong>Construct</strong></td><td class="col1">6.43 µg</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0"><strong>pCMV∆R8.74</strong></td><td class="col1">3.22 µg</td>
+
−
</tr>
+
−
<tr class="row6">
+
−
<td class="col0"><strong>pMD2G</strong></td><td class="col1">3.22 µg</td>
+
−
</tr>
+
−
<tr class="row7">
+
−
<td class="col0"><strong>N/P ration</strong></td><td class="col1">15</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_209'></div><div class="table sectionedit210"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Plasmid number</th><th class="col1">component</th><th class="col2">No. of Approaches</th><th class="col3">concentration[ng/µL]</th><th class="col4">volume [µL]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">pIG17_082</td><td class="col1"><strong>Mock(p526):EF1-GFP</strong></td><td class="col2">2</td><td class="col3">68</td><td class="col4">189.1</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">pIG17_114</td><td class="col1"><strong>Knock-down plasmid</strong></td><td class="col2">2</td><td class="col3">350; 432</td><td class="col4">18.4; 15</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">pIG17_115</td><td class="col1"><strong>Knock-down plasmid</strong></td><td class="col2">1</td><td class="col3">-</td><td class="col4">all two tubes</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">pIG17_116</td><td class="col1"><strong>Knock-down plasmid</strong></td><td class="col2">2</td><td class="col3">492; 356</td><td class="col4">20; 8.5</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">pIG17_003</td><td class="col1"><strong>Envelope plasmid pMD2G</strong></td><td class="col2">7</td><td class="col3">150;68.7;63.7</td><td class="col4">30;180;80</td>
+
−
</tr>
+
−
<tr class="row6">
+
−
<td class="col0">pIG17_004</td><td class="col1"><strong>Packaging Plasmid pCMV∆R8.74</strong></td><td class="col2">7</td><td class="col3">926.6</td><td class="col4">24.3</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_210'></div><ul>
+
−
<li class="level1"><div class="li"> Packaging Mastermix</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit211"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Component</th><th class="col1">7x [µL]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">pIG17_003</td><td class="col1">290</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">pIG17_004</td><td class="col1">24.3</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">150mM NaCl</td><td class="col1">3090.43</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_211'></div>
+
−
<p>
+
−
<strong>Follow our protocol of PEI transfection</strong>
+
−
</p>
+
−
<ol>
+
−
<li class="level1"><div class="li">  DNA:PEI ratio = 1:8</div>
+
−
</li>
+
−
<li class="level1"><div class="li">  Mix DNA with 1 ml serum free DMEM</div>
+
−
</li>
+
−
<li class="level1"><div class="li">  Add PEI while vortexing</div>
+
−
</li>
+
−
<li class="level1"><div class="li">  15 min incubation</div>
+
−
</li>
+
−
</ol>
+
−
<div class="table sectionedit212"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Component</th><th class="col1">Mass/approach[µg]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">pIG17_003</td><td class="col1">3.2</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">pIG17_004</td><td class="col1">3.2</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">pIG17_119</td><td class="col1">6.4</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">pIG17_121</td><td class="col1">6.4</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_212'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_208'></div>
+
−
<h2 class="sectionedit213" id="section030817">03.08.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_213'></div>
+
−
<h3 class="sectionedit214" id="hypoxia_-_seap_assay_in_hek_cells">Hypoxia - SEAP assay in HEK cells</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
- gather 200 µl of cell supernatant <br/>
+
−
 
+
−
- incubate cell supernatant at 65°C for 30min <br/>
+
−
 
+
−
- centrifuge cell supernatant for 1 min at 1250g <br/>
+
−
 
+
−
- add 80 µl supernatant into a 96 well plate <br/>
+
−
 
+
−
- add 100 µl 2x SEAP buffer <br/>
+
−
 
+
−
- add 20 µl pNPP and remove bubbles carefully <br/>
+
−
 
+
−
- measure in the plate reader every 30s for 2h <br/>
+
−
 
+
−
</p>
+
−
 
+
−
<p>
+
−
–&gt; Experiment failed and will be repeated
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_214'></div>
+
−
<h2 class="sectionedit215" id="section050817">05.08.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_215'></div>
+
−
<h3 class="sectionedit216" id="lentiviral_transduction5">Lentiviral Transduction</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Plate 200000 Jurkat cells/well in 6-well plate</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Titer Test of virus</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Incubation during weekend</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_216'></div>
+
−
<h3 class="sectionedit217" id="puromycin_killing_curve">Puromycin killing curve</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> testing of untransfected JK cells and the scramble JK cells that have been transfected with the KO Kit (for each 5 conditions will be tested)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> dilution of the Puro stock (10 mg/ml) 1:100 –&gt; 100 µg/ml</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> cells will be distributed in a 12 well plate with 1 ml of media</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit218"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">concentration of Puro</th><th class="col1">volume from 100 µg/ml [µl]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">0</td><td class="col1">0</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">0.125</td><td class="col1">1.25</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">0.2</td><td class="col1">2</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">0.25</td><td class="col1">2.5</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">0.5</td><td class="col1">5</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_218'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_217'></div>
+
−
<h2 class="sectionedit219" id="section060817">06.08.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_219'></div>
+
−
<h3 class="sectionedit220" id="pei_for_hre-seap_and_cmv-seaprepetition_of_the_seap_assay">PEI for HRE-SEAP and CMV-SEAP; repetition of the SEAP assay</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> cells will be distributed in a 12 well plate on the next day</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> one master mix for PEI</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 1 ml DMEM, 30 µl PEI, 10 µg DNA per each approach</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> cotransfection of CMV-SEAP (95%) and HRE-SEAP (95%) with CMV-GFP (5%)</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit221"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">construct</th><th class="col1">concentration [ng/µl]</th><th class="col2">volume added for 9.5 µg [µl]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">HRE-SEAP</td><td class="col1">248.2</td><td class="col2">38.3</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">CMV-SEAP</td><td class="col1">415.2</td><td class="col2">22.9</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_221'></div><div class="table sectionedit222"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">construct</th><th class="col1">concentration [ng/µl]</th><th class="col2">volume added for 0.5 µg [µl]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">CMV-GFP</td><td class="col1">303.1</td><td class="col2">1.6</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_222'></div><ul>
+
−
<li class="level1"><div class="li"> Per Approach: 9µg DNA, 2 mio JK cells</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_220'></div>
+
−
<h3 class="sectionedit223" id="biorad_electroporation15">BioRad Electroporation</h3>
+
−
<div class="level3">
+
−
<div class="table sectionedit224"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Approach</th><th class="col1">plasmid</th><th class="col2">Capacitance[µF]</th><th class="col3">Voltage</th><th class="col4">Resistance</th><th class="col5">Time Constance [msec]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1</td><td class="col1">no Plasmid</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2</td><td class="col1">CMV-GFP</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">3</td><td class="col1">CMV-GFP</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">4</td><td class="col1">Ca construct</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">5</td><td class="col1">Ca construct</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row6">
+
−
<td class="col0">6</td><td class="col1">Ca construct</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row7">
+
−
<td class="col0">7</td><td class="col1">Cas9-GFP-gRNA1</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row8">
+
−
<td class="col0">8</td><td class="col1">Cas9-GFP-gRNA1</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row9">
+
−
<td class="col0">9</td><td class="col1">Cas9-GFP-gRNA4</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row10">
+
−
<td class="col0">10</td><td class="col1">Cas9-GFP-gRNA4</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row11">
+
−
<td class="col0">11</td><td class="col1">Cas9-GFP-gRNA5</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row12">
+
−
<td class="col0">12</td><td class="col1">Cas9-GFP-gRNA5</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row13">
+
−
<td class="col0">13</td><td class="col1">Cas9-GFP-scramble</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row14">
+
−
<td class="col0">14</td><td class="col1">Cas9-GFP-scramble</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_224'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_223'></div>
+
−
<h2 class="sectionedit225" id="section070817">07.08.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_225'></div>
+
−
<h3 class="sectionedit226" id="pei_for_hre-seap_and_cmv-seaprepetition_of_the_seap_assay1">PEI for HRE-SEAP and CMV-SEAP; repetition of the SEAP assay</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> GFP was observed under the microscope</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> the cells were evenly distributed onto 12 well plates</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> induction with CoCl2 after the cells recovered from the transfer</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 8 conditions per approach, everything done in triplicates</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit227"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">concentration of CoCl2 [µM]</th><th class="col1">volume added [µl]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">0</td><td class="col1">0</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">100</td><td class="col1">0,5</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">150</td><td class="col1">0,75</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">200</td><td class="col1">1</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">250</td><td class="col1">1,25</td>
+
−
</tr>
+
−
<tr class="row6">
+
−
<td class="col0">300</td><td class="col1">1,5</td>
+
−
</tr>
+
−
<tr class="row7">
+
−
<td class="col0">500</td><td class="col1">2,5</td>
+
−
</tr>
+
−
<tr class="row8">
+
−
<td class="col0">1000</td><td class="col1">5</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_227'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_226'></div>
+
−
<h2 class="sectionedit228" id="section080817">08.08.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_228'></div>
+
−
<h3 class="sectionedit229" id="lentivirus_production_in_hek_cellsmock_pig17_133_pig17_119_pig17_121">Lentivirus production in HEK cells: Mock, pIG17_133, pIG17_119, pIG17_121</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
<a href="/igem2017/lib/exe/fetch.php?media=labor:pei_lentivirus_production_08.08.17.xlsx" class="media mediafile mf_xlsx" title="labor:pei_lentivirus_production_08.08.17.xlsx (41.9 KB)">pei_lentivirus_production_08.08.17.xlsx</a>
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_229'></div>
+
−
<h3 class="sectionedit230" id="pei_for_hre-seap_and_cmv-seaprepetition_of_the_seap_assay2">PEI for HRE-SEAP and CMV-SEAP; repetition of the SEAP assay</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
The SEAP assay was negative. No changes in the OD for either CMV-SEAP or HRE-SEAP could be observed. Due to these troubles the next test will be postponed till all constructs have undergone another test digestion and sequencing.
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_230'></div>
+
−
<h2 class="sectionedit231" id="section100817">10.08.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_231'></div>
+
−
<h3 class="sectionedit232" id="lentiviral_transduction6">Lentiviral transduction</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Virus: pIG17_082, pIG17_119, pIG17_121 and pIG17_133</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Plate 200000 virus for infection</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> As control: HEK with 1:1 infection</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_232'></div>
+
−
<h3 class="sectionedit233" id="cell_sortingko-plasmid_transfected_cells">Cell Sorting: KO-plasmid transfected cells</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> <strong>Cas9-GFP-scramble</strong></div>
+
−
</li>
+
−
</ul>
+
−
<img src="https://static.igem.org/mediawiki/2017/e/e2/T%E2%80%93FREIBURG%E2%80%9310.08.17_cell_sorting_KO_plasmid_transfected_cells_1.png">
+
−
<p>
+
−
<a href="/igem2017/lib/exe/detail.php?id=labor%3Acell_culture&amp;media=labor:20170810_jk_scramble.jpg" class="media" title="labor:20170810_jk_scramble.jpg"><img src="/igem2017/lib/exe/fetch.php?media=labor:20170810_jk_scramble.jpg" class="media" alt="" /></a>
+
−
</p>
+
−
<ul>
+
−
<li class="level1"><div class="li"> <strong>Cas9-GFP-gRNA1</strong></div>
+
−
</li>
+
−
</ul>
+
−
<img src="https://static.igem.org/mediawiki/2017/9/97/T%E2%80%93FREIBURG%E2%80%9310.08.17_cell_sorting_KO_plasmid_transfected_cells_2.png">
+
−
<p>
+
−
<a href="/igem2017/lib/exe/detail.php?id=labor%3Acell_culture&amp;media=labor:20170810_jk_grna_1.jpg" class="media" title="labor:20170810_jk_grna_1.jpg"><img src="/igem2017/lib/exe/fetch.php?media=labor:20170810_jk_grna_1.jpg" class="media" alt="" /></a>
+
−
</p>
+
−
<ul>
+
−
<li class="level1"><div class="li"> <strong>Cas9-GFP-gRNA4</strong></div>
+
−
</li>
+
−
</ul>
+
−
<img src="https://static.igem.org/mediawiki/2017/2/2d/T%E2%80%93FREIBURG%E2%80%9310.08.17_cell_sorting_KO_plasmid_transfected_cells_3.png">
+
−
<p>
+
−
<a href="/igem2017/lib/exe/detail.php?id=labor%3Acell_culture&amp;media=labor:20170810_jk_grna_4.jpg" class="media" title="labor:20170810_jk_grna_4.jpg"><img src="/igem2017/lib/exe/fetch.php?media=labor:20170810_jk_grna_4.jpg" class="media" alt="" /></a>
+
−
</p>
+
−
<ul>
+
−
<li class="level1"><div class="li"> <strong>Cas9-GFP-gRNA5</strong></div>
+
−
</li>
+
−
</ul>
+
−
<img src="https://static.igem.org/mediawiki/2017/d/df/T%E2%80%93FREIBURG%E2%80%9310.08.17_cell_sorting_KO_plasmid_transfected_cells_4.png">
+
−
<p>
+
−
<a href="/igem2017/lib/exe/detail.php?id=labor%3Acell_culture&amp;media=labor:20170810_jk_grna_5.jpg" class="media" title="labor:20170810_jk_grna_5.jpg"><img src="/igem2017/lib/exe/fetch.php?media=labor:20170810_jk_grna_5.jpg" class="media" alt="" /></a>
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_233'></div>
+
−
<h2 class="sectionedit234" id="section110817">11.08.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_234'></div>
+
−
<h3 class="sectionedit235" id="biorad_electroporation16">BioRad Electroporation</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> 9µg DNA and 2 mio. cells/ approach</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit236"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Approach</th><th class="col1">plasmid</th><th class="col2">Capacitance[µF]</th><th class="col3">Voltage</th><th class="col4">Resistance</th><th class="col5">Time Constance [msec]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1</td><td class="col1">no Plasmid</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2</td><td class="col1">KO Kit 203+101</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">3</td><td class="col1">KO Kit 203+104</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">4</td><td class="col1">Lenti-Cas9-Puro</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_236'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_235'></div>
+
−
<h3 class="sectionedit237" id="puromycin_killing_curve1">Puromycin killing curve</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
Day 6 after addition of puromycin: due to low cell numbers, the cells were spun down, resuspended in 100 µl and then counted. <br/>
+
−
 
+
−
</p>
+
−
 
+
−
<p>
+
−
Results for untransfected Jurkat cells and Jurkat cells transfected with the scramble:
+
−
</p>
+
−
<img src="https://static.igem.org/mediawiki/2017/d/d1/T%E2%80%93FREIBURG%E2%80%9311.08.17_puromycin_killing_curve_1.png">
+
−
<img src="https://static.igem.org/mediawiki/2017/8/8a/T%E2%80%93FREIBURG%E2%80%9311.08.17_puromycin_killing_curve_2.png">
+
−
<p>
+
−
<a href="/igem2017/lib/exe/detail.php?id=labor%3Acell_culture&amp;media=labor:screen_shot_2017-08-16_at_20.43.01.png" class="media" title="labor:screen_shot_2017-08-16_at_20.43.01.png"><img src="/igem2017/lib/exe/fetch.php?media=labor:screen_shot_2017-08-16_at_20.43.01.png" class="media" alt="" /></a>
+
−
<a href="/igem2017/lib/exe/detail.php?id=labor%3Acell_culture&amp;media=labor:screen_shot_2017-08-16_at_20.43.10.png" class="media" title="labor:screen_shot_2017-08-16_at_20.43.10.png"><img src="/igem2017/lib/exe/fetch.php?media=labor:screen_shot_2017-08-16_at_20.43.10.png" class="media" alt="" /></a>
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_237'></div>
+
−
<h3 class="sectionedit238" id="puromycin_addition_to_the_electroplated_ko_kit_cells">Puromycin addition to the electroplated KO Kit cells</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
Approaches:
+
−
</p>
+
−
<ul>
+
−
<li class="level1"><div class="li"> 1x scramble no treatment</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 1x scramble 0.2 µg/ml</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 2x KO Kit 203+101 0.2 µg</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 2x KO Kit 203+104 0.2 µg</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
<p>
+
−
Transfer to a 6 well plate
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_238'></div>
+
−
<h2 class="sectionedit239" id="section120817">12.08.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_239'></div>
+
−
<h3 class="sectionedit240" id="puromycinelectroplated_ko_kit_cells">Puromycin: electroplated KO Kit cells</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
Due to low cell counts the duplicates were pooled together and the cells transferred to a 12 well plate.
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_240'></div>
+
−
<h2 class="sectionedit241" id="section140817">14.08.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_241'></div>
+
−
<h3 class="sectionedit242" id="peihre-seap_pww56">PEI: HRE-SEAP, pWW56</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
in 10cm plates: <br/>
+
−
 
+
−
</p>
+
−
<ul>
+
−
<li class="level1"><div class="li"> 10 µg DNA</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 1ml DMEM</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 30 µl PEI</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
<p>
+
−
approaches: <br/>
+
−
 
+
−
</p>
+
−
<ul>
+
−
<li class="level1"><div class="li"> HRE-SEAP</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> pWW56 as a positive SEAP control</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> untransfected control</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit243"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">construct</th><th class="col1">concentration [ng/µl]</th><th class="col2">volume used for 10 µg DNA</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">HRE-SEAP</td><td class="col1">420,5</td><td class="col2">23,78</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">pWW56</td><td class="col1">555,5</td><td class="col2">18,00</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_243'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_242'></div>
+
−
<h2 class="sectionedit244" id="section150817">15.08.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_244'></div>
+
−
<h3 class="sectionedit245" id="seap">SEAP</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> the cells were evenly distributed onto 12 well plates</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> induction with CoCl2 after the cells recovered from the transfer</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 8 conditions per approach, everything done in triplicates</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit246"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">concentration of CoCl2 [µM]</th><th class="col1">volume added [µl]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">0</td><td class="col1">0</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">100</td><td class="col1">0,5</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">150</td><td class="col1">0,75</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">200</td><td class="col1">1</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">250</td><td class="col1">1,25</td>
+
−
</tr>
+
−
<tr class="row6">
+
−
<td class="col0">300</td><td class="col1">1,5</td>
+
−
</tr>
+
−
<tr class="row7">
+
−
<td class="col0">500</td><td class="col1">2,5</td>
+
−
</tr>
+
−
<tr class="row8">
+
−
<td class="col0">1000</td><td class="col1">5</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_246'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_245'></div>
+
−
<h3 class="sectionedit247" id="electroporated_ko_kit_cells">Electroporated KO Kit cells</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
Due to low cell counts the samples were transferred to a conical 96-well plate
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_247'></div>
+
−
<h3 class="sectionedit248" id="biorad_electroporation17">BioRad Electroporation</h3>
+
−
<div class="level3">
+
−
<div class="table sectionedit249"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Approach</th><th class="col1">plasmid</th><th class="col2">Capacitance[µF]</th><th class="col3">Voltage</th><th class="col4">Resistance</th><th class="col5">Time Constance [msec]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">1</td><td class="col1">no Plasmid</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2</td><td class="col1">CMV-GFP + CMV-mCherry</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">3</td><td class="col1">pIG17_086</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">4</td><td class="col1">pIG17_086</td><td class="col2">950</td><td class="col3">250</td><td class="col4">∞</td><td class="col5">20</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_249'></div>
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_248'></div>
+
−
<h3 class="sectionedit250" id="lentivirus_production_in_hek_cells">Lentivirus Production in HEK cells</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Approached by Frederike (AG Schamel)</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 2 Approaches (pIG17_119 &amp; pIG17_121) with our HEK cells, envelope &amp; packaging plasmid,PEI</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 1 Approach (pIG17_119) with material from AG Schamel: HEK, envelope &amp; packaging plasmid, PEI</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_250'></div>
+
−
<h2 class="sectionedit251" id="section160817">16.08.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_251'></div>
+
−
<h3 class="sectionedit252" id="seap1">SEAP</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
The SEAP assay was performed as mentioned before, however, there was a 1:5 dilution included of the HRE-SEAP samples.<br/>
+
−
 
+
−
</p>
+
−
 
+
−
<p>
+
−
For the analysis of the SEAP assay, the averages of the triplicates were calculated and the respective wild-type control subtracted from the HRE-SEAP samples. <br/>
+
−
 
+
−
</p>
+
−
 
+
−
<p>
+
−
For the undiluted HRE-SEAP construct the following graph was obtained:
+
−
<a href="/igem2017/lib/exe/detail.php?id=labor%3Acell_culture&amp;media=labor:screen_shot_2017-10-17_at_15.36.04.png" class="media" title="labor:screen_shot_2017-10-17_at_15.36.04.png"><img src="/igem2017/lib/exe/fetch.php?media=labor:screen_shot_2017-10-17_at_15.36.04.png" class="media" alt="" /></a>
+
−
</p>
+
−
<img src="https://static.igem.org/mediawiki/2017/2/2d/T%E2%80%93FREIBURG%E2%80%9316.08.17_SEAP_1.png">
+
−
<p>
+
−
For the 1:5 dilution the following graph was obtained:
+
−
<a href="/igem2017/lib/exe/detail.php?id=labor%3Acell_culture&amp;media=labor:screen_shot_2017-10-17_at_15.39.50.png" class="media" title="labor:screen_shot_2017-10-17_at_15.39.50.png"><img src="/igem2017/lib/exe/fetch.php?media=labor:screen_shot_2017-10-17_at_15.39.50.png" class="media" alt="" /></a>
+
−
</p>
+
−
<img src="https://static.igem.org/mediawiki/2017/5/5d/T%E2%80%93FREIBURG%E2%80%9316.08.17_SEAP_2.png">
+
−
<p>
+
−
For the 1:5 dilution absorbance values are missing at the start because they were negative and therefore regarded as 0.
+
−
</p>
+
−
 
+
−
<p>
+
−
The positive control did not work due to the promoter of this construct which was dependent on an input that wasn&#039;t given.
+
−
Furthermore, the samples did not show what we expected, considering that the uninduced control showed a steeper slope than most of the samples. Due to this reason the experiment was repeated.<br/>
+
−
 
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_252'></div>
+
−
<h2 class="sectionedit253" id="section170817">17.08.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_253'></div>
+
−
<h3 class="sectionedit254" id="lentiviral_transduction_with_virus-medium">Lentiviral Transduction with virus-medium</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Approaches for Titer test: both normal transduction and spin-infection.</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> <strong>On 8/20</strong>: After washing the cells, there was no signal from reporter gene expression.</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_254'></div>
+
−
<h2 class="sectionedit255" id="section230817">23.08.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_255'></div>
+
−
<h3 class="sectionedit256" id="pei_transfection_of_hek_cells_for_lentivirus_production">PEI transfection of HEK cells for lentivirus production</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Transferplasmid: pIG17_130 (duplicate)</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
<p>
+
−
<a href="/igem2017/lib/exe/fetch.php?media=labor:transfection_pei_lentivirus_23.08.17.xlsx" class="media mediafile mf_xlsx" title="labor:transfection_pei_lentivirus_23.08.17.xlsx (17 KB)">transfection_pei_lentivirus_23.08.17.xlsx</a>
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_256'></div>
+
−
<h2 class="sectionedit257" id="section250817">25.08.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_257'></div>
+
−
<h3 class="sectionedit258" id="concentrate_virus_pig17_130">Concentrate virus (pIG17_130)</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Add 8 ml 20% Sucrose (in PBS + 1Mm EDTA) in 50 ml Falcon Tube</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Add virus media slowly over sucrose (Use 25ml pippet) </div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Centrifuge over night at 4000 rpm, 4°C </div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_258'></div>
+
−
<h2 class="sectionedit259" id="section260817">26.08.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_259'></div>
+
−
<h3 class="sectionedit260" id="lentiviral_transduction_pig17_130">Lentiviral Transduction (pIG17_130)</h3>
+
−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> Plate 200000 cells/well (HEK &amp; Jurkat) into 6 well plate</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Titer of virus: 0, 15 µl, 30 µl</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
<p>
+
−
<a href="/igem2017/lib/exe/detail.php?id=labor%3Acell_culture&amp;media=labor:hek_130_transduction.jpg" class="media" title="labor:hek_130_transduction.jpg"><img src="/igem2017/lib/exe/fetch.php?media=labor:hek_130_transduction.jpg" class="media" alt="" /></a><br/>
+
−
 
+
−
As shown in the picture the transduction of HEK cells showed high efficiency. The cells were also mycoplasma-negative, so that the cells can be sorted at Bioss.<br/>
+
−
 
+
−
</p>
+
−
 
+
−
<p>
+
−
<a href="/igem2017/lib/exe/detail.php?id=labor%3Acell_culture&amp;media=labor:jk_130_transduction_.jpg" class="media" title="labor:jk_130_transduction_.jpg"><img src="/igem2017/lib/exe/fetch.php?media=labor:jk_130_transduction_.jpg" class="media" alt="" /></a><br/>
+
−
 
+
−
The efficiency of transduction was quite low. The positive signal showed above may come from dead cells. New transduction was done on 8/30 with higher virus titer (45µl and 60µl). <br/>
+
−
 
+
−
</p>
+
−
 
+
−
<p>
+
−
<a href="/igem2017/lib/exe/detail.php?id=labor%3Acell_culture&amp;media=labor:lentiviral_transduction_jk_130_.jpg" class="media" title="labor:lentiviral_transduction_jk_130_.jpg"><img src="/igem2017/lib/exe/fetch.php?media=labor:lentiviral_transduction_jk_130_.jpg" class="media" alt="" /></a><br/>
+
−
 
+
−
Lentiviral transduction of Jurkat cells with higher virus titer showed higher efficiency than previous try. These cells are sent for microplasma-test and for cell sorting.<br/>
+
−
 
+
−
</p>
+
−
 
+
−
</div>
+
−
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<h2 class="sectionedit261" id="section300817">30.08.17</h2>
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<h3 class="sectionedit262" id="pei_transfection_of_hek_cells_for_lentivirus_production_133_134">PEI transfection of HEK cells for lentivirus production (133&amp;134)</h3>
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<a href="/igem2017/lib/exe/fetch.php?media=labor:pei_transfection_of_hek_virus_30.08.17.xlsx" class="media mediafile mf_xlsx" title="labor:pei_transfection_of_hek_virus_30.08.17.xlsx (16.9 KB)">pei_transfection_of_hek_virus_30.08.17.xlsx</a>
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<h2 class="sectionedit263" id="section020917">02.09.17</h2>
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<div class='secedit editbutton_section editbutton_263'></div>
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<h3 class="sectionedit264" id="pei_transfection_of_hek_cells_for_lentivirus_production_132">PEI transfection of HEK cells for lentivirus production (132)</h3>
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<div class="level3">
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<a href="/igem2017/lib/exe/fetch.php?media=labor:pei_transfection_of_hek_virus_2.09.17.xlsx" class="media mediafile mf_xlsx" title="labor:pei_transfection_of_hek_virus_2.09.17.xlsx (16.8 KB)">pei_transfection_of_hek_virus_2.09.17.xlsx</a>
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<div class="tags"><span>
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<a href="/igem2017/doku.php?id=tag:labbooks&amp;do=showtag&amp;tag=labbooks" class="wikilink1" title="tag:labbooks" rel="tag">labbooks</a>
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</span></div>
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<div class='secedit editbutton_section editbutton_264'></div>
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<h2 class="sectionedit265" id="section040917">04.09.17</h2>
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<div class="level2">
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<div class='secedit editbutton_section editbutton_265'></div>
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<h3 class="sectionedit266" id="biorad_electroporation18">BioRad Electroporation</h3>
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<div class="level3">
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<p>
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<strong>9µg for each approach</strong><br/>
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</p>
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<ul>
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<li class="level1"><div class="li"> Setting: 12 well plate, 1 well set, 1 pulse</div>
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</li>
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</ul>
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<div class="table sectionedit267"><table class="inline">
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<thead>
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<tr class="row0">
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<th class="col0">Number of approaches</th><th class="col1">Plasmid</th><th class="col2">Concentration [ng/µl]</th><th class="col3">Volume for 9ng [µl]</th><th class="col4">Capacitance[µF]</th><th class="col5">Voltage</th><th class="col6">Resistance</th><th class="col7">Time Constance [msec]</th>
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<tr class="row1">
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<td class="col0">1</td><td class="col1">no DNA</td><td class="col2">0</td><td class="col3">0</td><td class="col4">950</td><td class="col5">250</td><td class="col6">∞</td><td class="col7">20</td>
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</tr>
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<tr class="row2">
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<td class="col0">1</td><td class="col1">CMV GFP, CMV mCherry</td><td class="col2">327.1, 1200</td><td class="col3">1376, 3.75</td><td class="col4">950</td><td class="col5">250</td><td class="col6">∞</td><td class="col7">20</td>
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</tr>
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<tr class="row3">
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<td class="col0">2</td><td class="col1">pIG17_031</td><td class="col2">746.9</td><td class="col3">12.05</td><td class="col4">950</td><td class="col5">250</td><td class="col6">∞</td><td class="col7">20</td>
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</tr>
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<tr class="row4">
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<td class="col0">2</td><td class="col1">pIG17_034</td><td class="col2">1210.9</td><td class="col3">7.43</td><td class="col4">950</td><td class="col5">250</td><td class="col6">∞</td><td class="col7">20</td>
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</tr>
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<tr class="row5">
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<td class="col0">2</td><td class="col1">pIG17_037</td><td class="col2">851.9</td><td class="col3">10.56</td><td class="col4">950</td><td class="col5">250</td><td class="col6">∞</td><td class="col7">20</td>
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<div class='secedit editbutton_section editbutton_266'></div>
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<h2 class="sectionedit268" id="section050917">05.09.17</h2>
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<div class="level2">
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<p>
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Washing of the electroporated cells
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</p>
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</div>
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<div class='secedit editbutton_section editbutton_268'></div>
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<h2 class="sectionedit269" id="section060917">06.09.17</h2>
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<div class="level2">
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<p>
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Sorting of the electroporated cells
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Gating after:
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</p>
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<ul>
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<li class="level1"><div class="li"> living cells</div>
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</li>
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<li class="level1"><div class="li"> single cells</div>
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</li>
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<li class="level1"><div class="li"> mCherry positive cells</div>
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</li>
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</ul>
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<p>
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Sorting strategy: <br/>
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−
 
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−
Control <a href="/igem2017/lib/exe/fetch.php?media=labor:igem_gfp_mcherry_jurkat_elektro_tube_001_06092017102918.pdf" class="media mediafile mf_pdf" title="labor:igem_gfp_mcherry_jurkat_elektro_tube_001_06092017102918.pdf (31.9 KB)">igem_gfp_mcherry_jurkat_elektro_tube_001_06092017102918.pdf</a> <br/>
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pIG17_031 <a href="/igem2017/lib/exe/fetch.php?media=labor:igem_gfp_mcherry_jurkat_31_tube_001_06092017103019.pdf" class="media mediafile mf_pdf" title="labor:igem_gfp_mcherry_jurkat_31_tube_001_06092017103019.pdf (35.4 KB)">igem_gfp_mcherry_jurkat_31_tube_001_06092017103019.pdf</a> <br/>
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pIG17_034 <a href="/igem2017/lib/exe/fetch.php?media=labor:igem_gfp_mcherry_jurkat_34_tube_001_06092017110102.pdf" class="media mediafile mf_pdf" title="labor:igem_gfp_mcherry_jurkat_34_tube_001_06092017110102.pdf (34.3 KB)">igem_gfp_mcherry_jurkat_34_tube_001_06092017110102.pdf</a> <br/>
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pIG17_037 <a href="/igem2017/lib/exe/fetch.php?media=labor:igem_gfp_mcherry_jurkat_37_tube_001_06092017112901.pdf" class="media mediafile mf_pdf" title="labor:igem_gfp_mcherry_jurkat_37_tube_001_06092017112901.pdf (35 KB)">igem_gfp_mcherry_jurkat_37_tube_001_06092017112901.pdf</a> <br/>
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<br/>
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Sorting reports: <br/>
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130 Jurkat: <a href="/igem2017/lib/exe/fetch.php?media=labor:sort_report_06092017122940.pdf" class="media mediafile mf_pdf" title="labor:sort_report_06092017122940.pdf (23 KB)">sort_report_06092017122940.pdf</a> <br/>
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pIG17_034 <a href="/igem2017/lib/exe/fetch.php?media=labor:sort_report_06092017112206.pdf" class="media mediafile mf_pdf" title="labor:sort_report_06092017112206.pdf (23.3 KB)">sort_report_06092017112206.pdf</a> <br/>
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pIG17_037 <a href="/igem2017/lib/exe/fetch.php?media=labor:sort_report_06092017120252.pdf" class="media mediafile mf_pdf" title="labor:sort_report_06092017120252.pdf (23.2 KB)">sort_report_06092017120252.pdf</a> <br/>
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</div>
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<div class='secedit editbutton_section editbutton_269'></div>
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<h2 class="sectionedit270" id="section070917">07.09.17</h2>
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<div class="level2">
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<p>
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−
Induction of the promoters
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</p>
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</div>
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<div class='secedit editbutton_section editbutton_270'></div>
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<h3 class="sectionedit271" id="set-up_for_pig17_031_-_hypoxia">Set-up for pIG17_031 - hypoxia</h3>
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<div class="level3">
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−
 
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<p>
+
−
- in a 96 well plate with 200 µl volume:
+
−
</p>
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−
<ul>
+
−
<li class="level1"><div class="li"> used concentration of CoCl2: 20 mM</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> approximately 15000 cells per approach </div>
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−
</li>
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−
</ul>
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−
<div class="table sectionedit272"><table class="inline">
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<thead>
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<tr class="row0">
+
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<th class="col0">CoCl2 concentration [µM]</th><th class="col1">Number of approaches</th><th class="col2">Volume added [µl]</th>
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</tr>
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−
</thead>
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−
<tr class="row1">
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−
<td class="col0">0</td><td class="col1">1</td><td class="col2">0</td>
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−
</tr>
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−
<tr class="row2">
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−
<td class="col0">50</td><td class="col1">2</td><td class="col2">0.5</td>
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</tr>
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−
<tr class="row3">
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<td class="col0">100</td><td class="col1">2</td><td class="col2">1</td>
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−
</tr>
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−
<tr class="row4">
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−
<td class="col0">200</td><td class="col1">2</td><td class="col2">2</td>
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−
</tr>
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−
<tr class="row5">
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−
<td class="col0">400</td><td class="col1">2</td><td class="col2">4</td>
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−
</tr>
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−
<tr class="row6">
+
−
<td class="col0">800</td><td class="col1">2</td><td class="col2">8</td>
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−
</tr>
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</table></div>
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<div class='secedit editbutton_table editbutton_272'></div>
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<p>
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.
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</p>
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−
 
+
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</div>
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<div class='secedit editbutton_section editbutton_271'></div>
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<h3 class="sectionedit273" id="set-up_for_pig17_034_-_ph">Set-up for pIG17_034 - pH</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
- in a 96 well plate with 200 µl volume:
+
−
</p>
+
−
<ul>
+
−
<li class="level1"><div class="li"> used concentration of NaOH: 1 M</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> used concentration of lactate: 0.6 M</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> used concentration of Forskolin: 10 mM</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> approximately 15000 cells per approach</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit274"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">Lactate (L), NaOH (N) or Forskolin (F) concentration or pH</th><th class="col1">Number of approaches</th><th class="col2">Volume added [µl]</th>
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−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">100 µM (F)</td><td class="col1">1</td><td class="col2">2</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">8.17 pH (N)</td><td class="col1">2</td><td class="col2">0.4</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">7.85 pH</td><td class="col1">2</td><td class="col2">0</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">6.54 pH (L)</td><td class="col1">2</td><td class="col2">8</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">6.19 pH (L)</td><td class="col1">2</td><td class="col2">10.67</td>
+
−
</tr>
+
−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_274'></div><ul>
+
−
<li class="level1"><div class="li"> 6 well plate with 3ml RPMI and the same concentration of lactate or NaOH as in the 96 well plate but without cells</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> used in order to measure the pH </div>
+
−
</li>
+
−
<li class="level1"><div class="li"> amount of cells will be neglected as it is rather low</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_273'></div>
+
−
<h3 class="sectionedit275" id="set-up_for_pig17_037_-_vegf">Set-up for pIG17_037 - VEGF</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
- in a 96 well plate with 200 µl volume:
+
−
</p>
+
−
<ul>
+
−
<li class="level1"><div class="li"> used concentration of VEGF: 1 µg/ml</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> approximately 15000 cells per approach</div>
+
−
</li>
+
−
</ul>
+
−
<div class="table sectionedit276"><table class="inline">
+
−
<thead>
+
−
<tr class="row0">
+
−
<th class="col0">VEGF concentration [ng/ml]</th><th class="col1">Number of approaches</th><th class="col2">Volume added [µl]</th>
+
−
</tr>
+
−
</thead>
+
−
<tr class="row1">
+
−
<td class="col0">0</td><td class="col1">1</td><td class="col2">0</td>
+
−
</tr>
+
−
<tr class="row2">
+
−
<td class="col0">2.5</td><td class="col1">2</td><td class="col2">0.5</td>
+
−
</tr>
+
−
<tr class="row3">
+
−
<td class="col0">5</td><td class="col1">2</td><td class="col2">1</td>
+
−
</tr>
+
−
<tr class="row4">
+
−
<td class="col0">10</td><td class="col1">2</td><td class="col2">2</td>
+
−
</tr>
+
−
<tr class="row5">
+
−
<td class="col0">20</td><td class="col1">2</td><td class="col2">4</td>
+
−
</tr>
+
−
<tr class="row6">
+
−
<td class="col0">40</td><td class="col1">2</td><td class="col2">8</td>
+
−
</tr>
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−
</table></div>
+
−
<div class='secedit editbutton_table editbutton_276'></div>
+
−
</div>
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−
<div class='secedit editbutton_section editbutton_275'></div>
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<h2 class="sectionedit277" id="section270817070917">27.08.17 - 07.09.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_277'></div>
+
−
<h3 class="sectionedit278" id="neomycin_killing_curve_of_jurkat_cells">Neomycin killing curve of Jurkat cells</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
<a href="/igem2017/lib/exe/detail.php?id=labor%3Acell_culture&amp;media=labor:neomycin_killing_curve_jk.png" class="media" title="labor:neomycin_killing_curve_jk.png"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=d52001&amp;media=labor:neomycin_killing_curve_jk.png" class="media" alt="" width="400" /></a>
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_278'></div>
+
−
<h2 class="sectionedit279" id="section110917">11.09.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_279'></div>
+
−
<h3 class="sectionedit280" id="pei_transfection_of_hek_cells_for_lenvirus_production">PEI transfection of HEK cells for lenvirus production</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
<a href="/igem2017/lib/exe/fetch.php?media=labor:transfection_pei_11.9.17.xlsx" class="media mediafile mf_xlsx" title="labor:transfection_pei_11.9.17.xlsx (16.9 KB)">transfection_pei_11.9.17.xlsx</a>
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_280'></div>
+
−
<h3 class="sectionedit281" id="viafect_-_new_testing">Viafect - new testing</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
Jurkat cells with CMV-GFP; transfection mix:
+
−
</p>
+
−
<ol>
+
−
<li class="level1"><div class="li"> 12 well plate</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 2 mio cells per well in 500 µl medium</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 0.5 µg DNA per well</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Viafect/DNA = 2:1 and 3:1</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> 50 µl total volume per approach (filled up with serum free DMEM)</div>
+
−
</li>
+
−
</ol>
+
−
<ul>
+
−
<li class="level1"><div class="li"> removal of the medium from pelleted cells</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> resuspending of pelleted cells in the transfection  mix</div>
+
−
</li>
+
−
<li class="level1 node"><div class="li"> addition of 500 µl RPMI 1649 with FCS after</div>
+
−
<ol>
+
−
<li class="level4"><div class="li"> 0 seconds</div>
+
−
</li>
+
−
<li class="level4"><div class="li"> 4 seconds</div>
+
−
</li>
+
−
<li class="level4"><div class="li"> 8 seconds</div>
+
−
</li>
+
−
<li class="level4"><div class="li"> 16 seconds</div>
+
−
</li>
+
−
<li class="level4"><div class="li"> 32 seconds</div>
+
−
</li>
+
−
</ol>
+
−
</li>
+
−
<li class="level1"><div class="li"> transfer of the cell suspension to a 24 well plate</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
<p>
+
−
Analysis in the fluorescence microscope showed that this method did not work again
+
−
</p>
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_281'></div>
+
−
<h2 class="sectionedit282" id="section150917">15.09.17</h2>
+
−
<div class="level2">
+
−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_282'></div>
+
−
<h3 class="sectionedit283" id="pei_transfection_of_hek_cells_for_lenvirus_production1">PEI transfection of HEK cells for lenvirus production</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
Constructs used: pIG17_130, CRE 4x <br/>
+
−
 
+
−
<a href="/igem2017/lib/exe/fetch.php?media=labor:transfection_pei.pdf" class="media mediafile mf_pdf" title="labor:transfection_pei.pdf (84 KB)">transfection_pei.pdf</a>
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<div class='secedit editbutton_section editbutton_283'></div>
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<h2 class="sectionedit284" id="section200917">20.09.17</h2>
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<div class="level2">
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<div class='secedit editbutton_section editbutton_284'></div>
+
−
<h3 class="sectionedit285" id="pei_transfection_of_hek_cells_for_lenvirus_production2">PEI transfection of HEK cells for lenvirus production</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
Constructs used: NFAT 4x, HRE 4x <br/>
+
−
 
+
−
<a href="/igem2017/lib/exe/fetch.php?media=labor:transfection_pei_20.9..pdf" class="media mediafile mf_pdf" title="labor:transfection_pei_20.9..pdf (924.4 KB)">transfection_pei_20.9..pdf</a>
+
−
The concentrated viruses were stored in -80°C freezer as backup.<br/>
+
−
 
+
−
</p>
+
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+
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<div class='secedit editbutton_section editbutton_285'></div>
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<h2 class="sectionedit286" id="section210917">21.09.17</h2>
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<div class="level2">
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</div>
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<div class='secedit editbutton_section editbutton_286'></div>
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<h3 class="sectionedit287" id="pei_transfection_of_hek_cells_for_seap_assay">PEI transfection of HEK cells for SEAP assay</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
The set up for the SEAP assay was the same as before, however there wild-type controls with the same amount of CoCl2 included.<br/>
+
−
 
+
−
All samples were done in triplicates<br/>
+
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+
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<div class='secedit editbutton_section editbutton_287'></div>
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<h2 class="sectionedit288" id="section220917">22.09.17</h2>
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<div class="level2">
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<div class='secedit editbutton_section editbutton_288'></div>
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<h3 class="sectionedit289" id="seap_assay">SEAP assay</h3>
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<div class="level3">
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+
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<p>
+
−
Induction with CoCl2, the used concentrations of CoCl2 were: 0, 20, 40, 80, 160, 320 and 640 µM.<br/>
+
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+
−
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+
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<div class='secedit editbutton_section editbutton_289'></div>
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<h2 class="sectionedit290" id="section230917">23.09.17</h2>
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<div class="level2">
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<div class='secedit editbutton_section editbutton_290'></div>
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<h3 class="sectionedit291" id="seap_assay1">SEAP assay</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
The SEAP assay was performed as before, this time we included a cell count of each pooled triplicate. <br/>
+
−
 
+
−
For the analysis first the averages of the triplicates were calculated and then divided by the cell count. Afterwards, the corresponding wild-type control was subtracted from the samples and all negative values were set to 0.
+
−
</p>
+
−
 
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−
<p>
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−
<a href="/igem2017/lib/exe/detail.php?id=labor%3Acell_culture&amp;media=labor:screen_shot_2017-10-17_at_15.55.43.png" class="media" title="labor:screen_shot_2017-10-17_at_15.55.43.png"><img src="/igem2017/lib/exe/fetch.php?media=labor:screen_shot_2017-10-17_at_15.55.43.png" class="media" alt="" /></a>
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−
</p>
+
−
 
+
−
<p>
+
−
We could see that only 20, 40 and 80 µM CoCl2 showed a steeper slope in our samples than the 0 µM control.
+
−
</p>
+
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</div>
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<div class='secedit editbutton_section editbutton_291'></div>
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<h2 class="sectionedit292" id="section240917">24.09.17</h2>
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<div class="level2">
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<div class='secedit editbutton_section editbutton_292'></div>
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<h3 class="sectionedit293" id="pei_transfection_of_hek_cells_for_lenvirus_production3">PEI transfection of HEK cells for lenvirus production</h3>
+
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<div class="level3">
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−
<p>
+
−
Constructs used: pIG17_134, CRE 4x <br/>
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<a href="/igem2017/lib/exe/fetch.php?media=labor:transfection_pei_lentivirus_24.9.17.xlsx" class="media mediafile mf_xlsx" title="labor:transfection_pei_lentivirus_24.9.17.xlsx (16.9 KB)">transfection_pei_lentivirus_24.9.17.xlsx</a>
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<div class='secedit editbutton_section editbutton_293'></div>
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<h2 class="sectionedit294" id="section2409171">24.09.17</h2>
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<div class='secedit editbutton_section editbutton_294'></div>
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<h3 class="sectionedit295" id="hre_testing_with_stable_hek_cell_lines">HRE testing with stable HEK cell lines</h3>
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<div class="level3">
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<h2 class="sectionedit296" id="section041017">04.10.17</h2>
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<div class="level2">
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<div class='secedit editbutton_section editbutton_296'></div>
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<h3 class="sectionedit297" id="pei_transfection_of_hek_cells_for_lenvirus_production4">PEI transfection of HEK cells for lenvirus production</h3>
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<div class="level3">
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−
<p>
+
−
Constructs used: pIG17_133, Suiside plasmid
+
−
<a href="/igem2017/lib/exe/fetch.php?media=labor:transfection_pei_4.10.17.xlsx" class="media mediafile mf_xlsx" title="labor:transfection_pei_4.10.17.xlsx (16.9 KB)">transfection_pei_4.10.17.xlsx</a>
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</p>
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<div class='secedit editbutton_section editbutton_297'></div>
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<h2 class="sectionedit298" id="section091017">09.10.17</h2>
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<div class="level2">
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</div>
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<div class='secedit editbutton_section editbutton_298'></div>
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<h3 class="sectionedit299" id="cfp_test">CFP test</h3>
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<div class="level3">
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<p>
+
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Due to the problems with our GFP readout we performed a search in BLAST and found out that our eGFP was wrongly annotate and is a eCFP. We also could see that in the FACS. For this we tested our Jurkat knockdown 130 cells, that stably express CFP then. On the left these cells are measured in the CFP channel and on the right in the GFP channel. The depicted cells are gated for living and single cells:<br/>
+
−
 
+
−
<a href="/igem2017/lib/exe/detail.php?id=labor%3Acell_culture&amp;media=labor:screen_shot_2017-10-12_at_11.53.16.png" class="media" title="labor:screen_shot_2017-10-12_at_11.53.16.png"><img src="/igem2017/lib/exe/fetch.php?media=labor:screen_shot_2017-10-12_at_11.53.16.png" class="media" alt="" /></a> <br/>
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−
 
+
−
</p>
+
−
 
+
−
<p>
+
−
There is a clear shift and our cells are really CFP positive and not GFP positive
+
−
</p>
+
−
 
+
−
</div>
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<div class='secedit editbutton_section editbutton_299'></div>
+
−
<h3 class="sectionedit300" id="cre_test_in_jurkat_cells">CRE Test in Jurkat cells</h3>
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−
<div class="level3">
+
−
 
+
−
<p>
+
−
<strong>With Forskolin and IBMX, the cAMP pathway in the cell is activated.</strong>
+
−
</p>
+
−
<ul>
+
−
<li class="level1"><div class="li"> Cell lines: WT Jurkat, stable CRE4X Jurkat</div>
+
−
</li>
+
−
<li class="level1"><div class="li"> Conditions: untreated, induction with 100 µM Forskolin, induction with 100 µM IBMX, induction with 100 µM Forskolin and 100 µM IBMX.</div>
+
−
</li>
+
−
</ul>
+
−
 
+
−
<p>
+
−
FACS: <br/>
+
−
 
+
−
Cells were gated for living, single, mCherry positive cells and then the amount of CFP positive cells was measured: <br/>
+
−
 
+
−
<a href="/igem2017/lib/exe/detail.php?id=labor%3Acell_culture&amp;media=labor:screen_shot_2017-10-17_at_17.01.57.png" class="media" title="labor:screen_shot_2017-10-17_at_17.01.57.png"><img src="/igem2017/lib/exe/fetch.php?media=labor:screen_shot_2017-10-17_at_17.01.57.png" class="media" alt="" /></a>
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</p>
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</div>
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<div class='secedit editbutton_section editbutton_300'>''</div>
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<h2 class="sectionedit301" id="section101017">10.10.17</h2>
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<div class="level2">
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</div>
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<div class='secedit editbutton_section editbutton_301'></div>
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<h3 class="sectionedit302" id="hre_test_in_jurkat_cells">HRE Test in Jurkat cells</h3>
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−
<div class="level3">
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−
 
+
−
</div>
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−
<div class='secedit editbutton_section editbutton_302'></div>
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<h3 class="sectionedit303" id="hre_test_in_hek_cells">HRE Test in HEK cells</h3>
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<div class="level3">
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−
 
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</div>
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<div class='secedit editbutton_section editbutton_303'></div>
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<h2 class="sectionedit304" id="section121017">12.10.17</h2>
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<div class="level2">
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</div>
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−
<div class='secedit editbutton_section editbutton_304'></div>
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<h3 class="sectionedit305" id="cre_test_in_hek_cells">CRE Test in HEK cells</h3>
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−
<div class="level3">
+
−
<ul>
+
−
<li class="level1"><div class="li"> On 11th of Oct.: PEI transfection with CMV-TDAG8 plasmid. </div>
+
−
</li>
+
−
</ul>
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−
 
+
−
</div>
+
−
<div class='secedit editbutton_section editbutton_305'></div>
+
−
<h3 class="sectionedit306" id="cre_test_in_jurkat_cells1">CRE Test in Jurkat cells</h3>
+
−
<div class="level3">
+
−
 
+
−
<p>
+
−
We obtained the following results after gating for living, single, mCherry positive cells:
+
−
<a href="/igem2017/lib/exe/detail.php?id=labor%3Acell_culture&amp;media=labor:screen_shot_2017-10-17_at_17.24.42.png" class="media" title="labor:screen_shot_2017-10-17_at_17.24.42.png"><img src="/igem2017/lib/exe/fetch.php?media=labor:screen_shot_2017-10-17_at_17.24.42.png" class="media" alt="" /></a>
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</p>
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−
</div>
+
−
<div class='secedit editbutton_section editbutton_306'></div>
+
−
<h3 class="sectionedit307" id="vegf_test_in_hek_cells">VEGF Test in HEK cells</h3>
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−
<div class="level3">
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−
 
+
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</div>
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−
<div class='secedit editbutton_section editbutton_307'></div>
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<div class="container">
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    <div class="row">
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      <div class="col-md-12 text-center">
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        <div class="flex-container">
+
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          <div class="item">
+
  
−
<h1 class="sectionedit1" id="lab_notebook_modeling">Lab Notebook Modeling</h1>
 
−
<div class="level1">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_1'> </div>
 
−
<h2 class="sectionedit2" id="section060617">06.06.17</h2>
 
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<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_2'> </div>
 
−
<h3 class="sectionedit3" id="titration_rpmi_1640_vs_lactic_acid">Titration RPMI 1640 vs. lactic acid</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<em class="u"><strong>Aim:</strong></em> setup of pH in the Medium for pCRE-tests<br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
stock-solutions:<br/>
 
−
 
−
c(lactic acid, conc.) = 12.09 mol/l<br/>
 
−
 
−
c(lactic acid, 1:20) = 0.6 mol/l (diluted with RPMI 1640)<br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
<em class="u"><strong>Execution:</strong></em>
 
−
</p>
 
−
<ol>
 
−
<li class="level1"><div class="li"> add lactic acid (0.6 M) to medium (Table)</div>
 
−
</li>
 
−
<li class="level2"><div class="li"> measure pH with pH-Meter</div>
 
−
</li>
 
−
<li class="level2"><div class="li"> repeat each measurement 3 times</div>
 
−
</li>
 
−
</ol>
 
−
<div class="table sectionedit4"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">V(RPMI 1640) [µl]</th><th class="col1">V(lactic acid, 0.6 M) [µl]</th><th class="col2">c(lactic acid) [µmol/mL]</th><th class="col3">s(c ) [µmol/ml]</th><th class="col4">pH 1</th><th class="col5">pH 2</th><th class="col6">pH 3</th><th class="col7">pH</th><th class="col8">s(pH)</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2000</td><td class="col1">20</td><td class="col2">5.99</td><td class="col3">0.2</td><td class="col4">7.14</td><td class="col5">7.18</td><td class="col6">7.16</td><td class="col7">7.16</td><td class="col8">0.01</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">2000</td><td class="col1">40</td><td class="col2">11.9</td><td class="col3">0.3</td><td class="col4">6.90</td><td class="col5">6.91</td><td class="col6">6.88</td><td class="col7">6.90</td><td class="col8">0.01</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">2000</td><td class="col1">60</td><td class="col2">17.6</td><td class="col3">0.5</td><td class="col4">6.57</td><td class="col5">6.61</td><td class="col6">6.66</td><td class="col7">6.61</td><td class="col8">0.03</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">2000</td><td class="col1">80</td><td class="col2">23.3</td><td class="col3">0.6</td><td class="col4">6.35</td><td class="col5">6.31</td><td class="col6">6.27</td><td class="col7">6.31</td><td class="col8">0.02</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">2000</td><td class="col1">100</td><td class="col2">28.8</td><td class="col3">0.8</td><td class="col4">5.91</td><td class="col5">5.65</td><td class="col6">5.75</td><td class="col7">5.77</td><td class="col8">0.08</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">2000</td><td class="col1">120</td><td class="col2">34.2</td><td class="col3">0.9</td><td class="col4">4.99</td><td class="col5">4.96</td><td class="col6">4.93</td><td class="col7">4.96</td><td class="col8">0.02</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0">2000</td><td class="col1">140</td><td class="col2">40</td><td class="col3">1</td><td class="col4">4.41</td><td class="col5">4.37</td><td class="col6">4.36</td><td class="col7">4.38</td><td class="col8">0.02</td>
 
−
</tr>
 
−
<tr class="row8">
 
−
<td class="col0">2000</td><td class="col1">160</td><td class="col2">45</td><td class="col3">1</td><td class="col4">4.31</td><td class="col5">4.12</td><td class="col6">4.10</td><td class="col7">4.18</td><td class="col8">0.07</td>
 
−
</tr>
 
−
<tr class="row9">
 
−
<td class="col0">2000</td><td class="col1">180</td><td class="col2">50</td><td class="col3">1</td><td class="col4">3.98</td><td class="col5">3.95</td><td class="col6">3.98</td><td class="col7">3.97</td><td class="col8">0.01</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_4'> </div>
 
−
<p>
 
−
<br/>
 
−
 
−
<em class="u"><strong>Resulting Titration curve for lactic acid in RPMI 1640:</strong></em><br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit5"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">Titration curve with pH vs. c(lactic acid)</th><th class="col1">Titration curve with pH vs. V(lactic acid 0.6 M) per ml</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0"><img src="https://static.igem.org/mediawiki/2017/1/18/T-Freiburg-6_6_2017_titration2.jpeg"><img src="https://static.igem.org/mediawiki/2017/e/ee/T-Freiburg-6_6_2017_titration.jpeg"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=titration_final.jpg" class="media" title="titration_final.jpg"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=b008f9&amp;media=titration_final.jpg" class="mediacenter" alt="" width="400" /></a></td><td class="col1"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=titration_final_volume.jpg" class="media" title="titration_final_volume.jpg"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=ab0a8c&amp;media=titration_final_volume.jpg" class="mediacenter" alt="" width="400" /></a></td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_5'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_3'> </div>
 
−
<h2 class="sectionedit6" id="section130617">13.06.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_6'> </div>
 
−
<h3 class="sectionedit7" id="cell_survival_and_cfp-stability_at_different_ph-values">Cell survival and CFP-stability at different pH-values</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
used cells: Jurkat with CMV_CFP<br/>
 
−
 
−
cell density: 1 mio cells/ml<br/>
 
−
 
−
incubation times: 3 h, 6 h, 12 h<br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
<em class="u"><strong>Execution:</strong></em><br/>
 
−
 
−
</p>
 
−
<ol>
 
−
<li class="level1"><div class="li"> count cells</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> centrifuge cells</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> resuspend cell pellet in RPMI 1640 (target-concentration: 2 mio cells/ml)</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> mix 1.5 ml RPMI 1640 + calculated volume of lactic acid (0.6 M) + 500 µl cell suspension on 12-well-plate (Table 1)</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> incubate for defined time in the CO<sub>2</sub>-incubator</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> centrifuge samples</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> resuspend cell pellets in PBS FACS buffer (500 µl)</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> FACS analysis</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> used medium was stored at 4°C for pH-test next day</div>
 
−
</li>
 
−
</ol>
 
−
<div class="table sectionedit8"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">pH</th><th class="col1">V(lactic acid, 0.6 M) [µl]</th><th class="col2">RPMI 1640 [µl]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">5.0</td><td class="col1">120</td><td class="col2">2000</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">5.5</td><td class="col1">107</td><td class="col2">2000</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">6.0</td><td class="col1">92</td><td class="col2">2000</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">6.5</td><td class="col1">68</td><td class="col2">2000</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">7.0</td><td class="col1">0</td><td class="col2">2000</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_8'> </div>
 
−
<p>
 
−
<br/>
 
−
 
−
<em class="u"><strong>Results:</strong></em><br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
→ Jurkat cells can survive in medium with pH ≥ 6.0<br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
<strong>cell survival</strong>
 
−
</p>
 
−
<div class="table sectionedit9"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">after 3h</th><th class="col1">after 6h</th><th class="col2">after 12h</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0"><img src="https://static.igem.org/mediawiki/2017/8/85/T-Freiburg-6_13_2017_cell_survival_CFP_stability_1.jpeg"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=photo_2017-06-14_15-15-20.jpg" class="media" title="photo_2017-06-14_15-15-20.jpg"><img src="/igem2017/lib/exe/fetch.php?w=300&amp;tok=78e39b&amp;media=photo_2017-06-14_15-15-20.jpg" class="mediacenter" alt="" width="300" /></a></td><td class="col1"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=photo_2017-06-14_15-15-36.jpg" class="media" title="photo_2017-06-14_15-15-36.jpg"><img src="/igem2017/lib/exe/fetch.php?w=300&amp;tok=9b5f60&amp;media=photo_2017-06-14_15-15-36.jpg" class="mediacenter" alt="" width="300" /></a></td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=photo_2017-06-14_15-15-42.jpg" class="media" title="photo_2017-06-14_15-15-42.jpg"><img src="/igem2017/lib/exe/fetch.php?w=300&amp;tok=291bdb&amp;media=photo_2017-06-14_15-15-42.jpg" class="mediacenter" alt="" width="300" /></a></td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_9'> </div>
 
−
<p>
 
−
<a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=cell_survival.jpg" class="media" title="cell_survival.jpg"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=01ecfc&amp;media=cell_survival.jpg" class="mediacenter" alt="" width="400" /></a>
 
−
</p>
 
−
 
−
<img src="https://static.igem.org/mediawiki/2017/0/0c/T-Freiburg_6_13_2017_cell_survival_CFP_stability_4.jpeg">
 
−
<p>
 
−
<strong>CFP fluorescence</strong>
 
−
</p>
 
−
<div class="table sectionedit10"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">after 3h</th><th class="col1">after 6h</th><th class="col2">after 12h</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0"><img src="https://static.igem.org/mediawiki/2017/a/a4/T-Freiburg-6_13_2017_cell_survival_CFP_stability_2.jpeg"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=photo_2017-06-14_15-15-48.jpg" class="media" title="photo_2017-06-14_15-15-48.jpg"><img src="/igem2017/lib/exe/fetch.php?w=300&amp;tok=9c7865&amp;media=photo_2017-06-14_15-15-48.jpg" class="mediacenter" alt="" width="300" /></a></td><td class="col1"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=photo_2017-06-14_15-15-56.jpg" class="media" title="photo_2017-06-14_15-15-56.jpg"><img src="/igem2017/lib/exe/fetch.php?w=300&amp;tok=17b2bf&amp;media=photo_2017-06-14_15-15-56.jpg" class="mediacenter" alt="" width="300" /></a></td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=photo_2017-06-14_15-16-01.jpg" class="media" title="photo_2017-06-14_15-16-01.jpg"><img src="/igem2017/lib/exe/fetch.php?w=300&amp;tok=1a7641&amp;media=photo_2017-06-14_15-16-01.jpg" class="mediacenter" alt="" width="300" /></a></td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_10'> </div>
 
−
<p>
 
−
<a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=fluorescence_intensity.jpg" class="media" title="fluorescence_intensity.jpg"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=c9ebab&amp;media=fluorescence_intensity.jpg" class="mediacenter" alt="" width="400" /></a>
 
−
</p>
 
−
 
−
<img src="https://static.igem.org/mediawiki/2017/d/d9/T%E2%80%93FREIBURG-6_13_2017_cell_survival_CFP_stability_8.jpeg">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_7'> </div>
 
−
<h2 class="sectionedit11" id="section140617">14.06.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_11'> </div>
 
−
<h3 class="sectionedit12" id="ph-measurement_of_used_medium_130617">pH-measurement of used medium (13.06.17)</h3>
 
−
<div class="level3">
 
−
<div class="table sectionedit13"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">set pH</th><th class="col1">6 h</th><th class="col2">12 h</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">pH = 5.0</td><td class="col1">4.67</td><td class="col2">4.78</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pH = 5.5</td><td class="col1">5.19</td><td class="col2">5.43</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">pH = 6.0</td><td class="col1">6.26</td><td class="col2">6.33</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">pH = 6.5</td><td class="col1">6.95</td><td class="col2">6.76</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">untreated (pH = 7.25)</td><td class="col1">7.73</td><td class="col2">7.47</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_13'> </div>
 
−
<p>
 
−
<strong>Result:</strong>
 
−
pH in incubation medium is rising over the time! Expected was a lowering, as the cells secrete lactic acid.
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_12'> </div>
 
−
<h2 class="sectionedit14" id="section170617">17.06.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_14'> </div>
 
−
<h3 class="sectionedit15" id="investigation_of_the_ph-changes">Investigation of the pH-changes</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
test of pH-change of RPMI 1640 at different conditions<br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
tested conditions:
 
−
</p>
 
−
<ul>
 
−
<li class="level1"><div class="li"> incubation in stock RPMI 1640 or RPMI 1640 with lactic acid (pH = 6.0)</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> medium with/without Jurkat cells (500k/ml)</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> incubation at 37°C in incubator with CO<sub>2</sub> (5%) atmosphere or incubated with cap</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> incubation at atmospheric conditions</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> incubation time: 6 h</div>
 
−
</li>
 
−
</ul>
 
−
<ol>
 
−
<li class="level1"><div class="li"> make aliquots of RPMI 1640 (15 ml, pH = 6.0) and untreated RPMI 1640 (15 ml)</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> aliquot 1: measure pH before incubation</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> other aliquots: incubate at conditions described above<br/>
 
−
</div>
 
−
</li>
 
−
</ol>
 
−
 
−
<p>
 
−
<strong>Results</strong>
 
−
</p>
 
−
<div class="table sectionedit16"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">6 h, 37 °C, 5% CO<sub>2</sub></th><th class="col1">RPMI 1640 (pH = 6.0)</th><th class="col2">stock RPMI 1640</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">pH before incubation</td><td class="col1">5.87</td><td class="col2">7.45</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pH after incubation</td><td class="col1">6.46</td><td class="col2">7.68</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_16'> </div>
 
−
<p>
 
−
<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit17"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">6 h, 37 °C, 5% CO<sub>2</sub>, Jurkat cells</th><th class="col1">RPMI 1640 (pH = 6.0)</th><th class="col2">stock RPMI 1640</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">pH before incubation</td><td class="col1">5.87</td><td class="col2">7.45</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pH after incubation</td><td class="col1">6.48</td><td class="col2">7.70</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_17'> </div>
 
−
<p>
 
−
<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit18"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">6 h, 37 °C, with cap</th><th class="col1">RPMI 1640 (pH = 6.0)</th><th class="col2">stock RPMI 1640</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">pH before incubation</td><td class="col1">5.87</td><td class="col2">7.45</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pH after incubation</td><td class="col1">6.63</td><td class="col2">8.46</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_18'> </div>
 
−
<p>
 
−
<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit19"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">6 h, 25 °C, atmospheric conditions</th><th class="col1">RPMI 1640 (pH = 6.0)</th><th class="col2">stock RPMI 1640</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">pH before incubation</td><td class="col1">5.87</td><td class="col2">7.45</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pH after incubation</td><td class="col1">6.24</td><td class="col2">7.79</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_19'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_15'> </div>
 
−
<h2 class="sectionedit20" id="section190617">19.06.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_20'> </div>
 
−
<h3 class="sectionedit21" id="ph_change_vs_incubation_time">pH change vs. incubation time</h3>
 
−
<div class="level3">
 
−
<ol>
 
−
<li class="level1"><div class="li"> set pH to 6.3</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> prepare 2 ml aliquots of RPMI 1640 in falcon tubes</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> incubate in CO<sub>2</sub> incubator for t (min) = 0, 10, 20, 30, 50</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> measure pH directly after taking sample out of incubator</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> measure pH one minute later</div>
 
−
</li>
 
−
</ol>
 
−
<div class="table sectionedit22"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">time /min</th><th class="col1">pH direct</th><th class="col2">pH after 1 min</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">0</td><td class="col1">6.32</td><td class="col2">6.32</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">10</td><td class="col1">6.03</td><td class="col2">6.25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">20</td><td class="col1">6.06</td><td class="col2">6.28</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">30</td><td class="col1">5.88</td><td class="col2">6.36</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">50</td><td class="col1">6.36</td><td class="col2">6.65</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_22'> </div>
 
−
<p>
 
−
pH is strongly rising after taking the sample out of the incubator. This may be caused by the solved CO<sub>2</sub>, that lowers the pH in the incubator. After taking the sample out of the incubator, the CO<sub>2</sub> goes to the gas phase which causes a rising of the pH. Additionally, the bicarbonate buffer is destabilized as the addition of lactic acid moves the equilibrium of the buffer to CO<sub>2</sub> + H<sub>2</sub>O. That leads to a higher pH than before the incubation.
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_21'> </div>
 
−
<h2 class="sectionedit23" id="section040717">04.07.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_23'> </div>
 
−
<h3 class="sectionedit24" id="vegf_dilution">VEGF dilution</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
m<sub>0</sub> = 10 µg
 
−
start concentration: solid state
 
−
target concentration: 1 µg/ml
 
−
</p>
 
−
<ol>
 
−
<li class="level1"><div class="li"> resuspend pellet in 100 µl ddH<sub>2</sub>O (stock solution, 100 µg/ml)</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> dilute 1 µl of stock solution with 99 µl ddH<sub>2</sub>O (aliquots, 1 µg/ml)</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> store aliquots at -20 °C</div>
 
−
</li>
 
−
</ol>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_24'> </div>
 
−
<h2 class="sectionedit25" id="section060717">06.07.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_25'> </div>
 
−
<h3 class="sectionedit26" id="pctla4_vs_vegf">pCTLA4 vs. [VEGF]</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>Aim:</strong> determination of suitable [VEGF] and incubation time<br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
<strong>tested cells</strong>: HEK with transient pCTLA4-CFP (pIG17_022 (330 bp); pIG17_023 (380 bp)),PEI transfected<br/>
 
−
 
−
[VEGF](ng/ml) = 0.0, 0.5, 5.0, 10.0<br/>
 
−
 
−
t (h) = 3, 8, 24<br/>
 
−
 
−
controls: CMV_CFP (pIG_009), untransfected cells
 
−
</p>
 
−
 
−
<p>
 
−
Execution:
 
−
</p>
 
−
<ol>
 
−
<li class="level1"><div class="li"> cells were PEI transfected by cell culture on 05.07.17</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> 24h after PEI transfection: replace old medium by RPMI 1640 (2 ml / well), add VEGF (1 µg/ml)</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> after incubation time: analysis via fluorescence microscope and FACS</div>
 
−
</li>
 
−
</ol>
 
−
 
−
<p>
 
−
<strong><em class="u">Results</em></strong>:
 
−
<a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=histogram_results.jpg" class="media" title="histogram_results.jpg"><img src="/igem2017/lib/exe/fetch.php?w=600&amp;tok=68fc80&amp;media=histogram_results.jpg" class="mediacenter" alt="" width="600" /></a>
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_26'> </div>
 
−
<h2 class="sectionedit27" id="section180717">18.07.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_27'> </div>
 
−
<h3 class="sectionedit28" id="pei_transfection_of_plasmids_pig17_022_23_37_86">PEI transfection of plasmids pIG17_022/23/37/86</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
used cells: HEK cells<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit29"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">Plasmid number</th><th class="col1">component</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">pIG17_022</td><td class="col1">Ctla4(380bp)-CFP</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pIG17_023</td><td class="col1">Ctla4(330bp)-CFP</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">pIG17_037</td><td class="col1">Ctla4(330bp)-CFP-CMV-mCherry</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">pIG17_086</td><td class="col1">Ctla4(380bp)-CFP-CMV-mCherry</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_29'> </div><ol>
 
−
<li class="level1"><div class="li"> Split the cells 1:5 into 10 cm plate (on 21.04.17)<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Thaw PEI reagent at RT<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> incubate 15 min at RT<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Add the mix to the culture<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> incubate for 24 h<br/>
 
−
</div>
 
−
</li>
 
−
</ol>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_28'> </div>
 
−
<h2 class="sectionedit30" id="section190717">19.07.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_30'> </div>
 
−
<h3 class="sectionedit31" id="vegf_test">VEGF test</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
used cells: PEI transfected cells from 19.07.17 with pIG17_022/023/037/086<br/>
 
−
 
−
</p>
 
−
<ol>
 
−
<li class="level1"><div class="li"> add following VEGF concentrations to the cells: 0 ng/ml, 0.5 ng/ml, 5 ng/ml, 10 ng/ml, 50 ng/ml<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> incubate for 24 h<br/>
 
−
</div>
 
−
</li>
 
−
</ol>
 
−
 
−
<p>
 
−
fluorescence microscope:<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit32"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">construct</th><th class="col1">bright field</th><th class="col2">CFP</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">positive control<br/>
 
−
CMV-CFP</td><td class="col1"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=gfp_pos_vis_black.png" class="media" title="gfp_pos_vis_black.png"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=64f251&amp;media=gfp_pos_vis_black.png" class="mediacenter" alt="" width="400" /></a></td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=gfp_pos_black.png" class="media" title="gfp_pos_black.png"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=70b3f6&amp;media=gfp_pos_black.png" class="mediacenter" alt="" width="400" /></a></td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">negative control</td><td class="col1"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=neg_vis_black.png" class="media" title="neg_vis_black.png"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=f5f53c&amp;media=neg_vis_black.png" class="mediacenter" alt="" width="400" /></a></td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=neg_black.png" class="media" title="neg_black.png"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=7a2af5&amp;media=neg_black.png" class="mediacenter" alt="" width="400" /></a></td>
 
−
</tr>
 
−
<tr class="row3">
 
−
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_32'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_31'> </div>
 
−
<h2 class="sectionedit33" id="section050817">05.08.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_33'> </div>
 
−
<h3 class="sectionedit34" id="interlab_study_transformation">Interlab Study transformation</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- heat shock transformation of DH5-α with plasmids from Kit plate 7<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit35"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">well</th><th class="col1">approach</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">21B</td><td class="col1">positive control</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">21D</td><td class="col1">negative control</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">21F</td><td class="col1">Device 1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">21H</td><td class="col1">Device 2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">21J</td><td class="col1">Device 3</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">21L</td><td class="col1">Device 4</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0">21N</td><td class="col1">Device 5</td>
 
−
</tr>
 
−
<tr class="row8">
 
−
<td class="col0">21P</td><td class="col1">Device 6</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_35'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_34'> </div>
 
−
<h3 class="sectionedit36" id="interlab_study_od600_calibration_measurements">Interlab Study OD600 calibration measurements</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- measure absorbance at 600 nm of H20 (100 µl) and LUDOX-S40 (100 µl)
 
−
- each four replicates<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit37"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">replicate</th><th class="col1">LUDOX-S40</th><th class="col2">H2O</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">1</td><td class="col1">0,043</td><td class="col2 rightalign"> 0,035</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">2</td><td class="col1">0,049</td><td class="col2 rightalign"> 0,036</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">3</td><td class="col1">0,047</td><td class="col2 rightalign"> 0,035</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">4</td><td class="col1">0,052</td><td class="col2 rightalign"> 0,035</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_37'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_36'> </div>
 
−
<h3 class="sectionedit38" id="interlab_study_fluorescein_standard_curve_measurements">Interlab Study fluorescein standard curve measurements</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- centrifuge fluorescein<br/>
 
−
 
−
- dilute fluorescein in PBS to a final concentration of 50 µM<br/>
 
−
 
−
- add PBS (100 µl) into well A2-A12, B2-B12, C2-C12, D2-D12 of a 96-well-plate<br/>
 
−
 
−
- add fluorescein stock solution (200 µl, 50µM) into well A1, B1, C1, D1<br/>
 
−
 
−
- transfer 100 µl from well A1 into well A2, pipett up and down<br/>
 
−
 
−
- transfer 100 µl from well A2 into well A3, pipett up and down<br/>
 
−
 
−
- do the same till the end of the row<br/>
 
−
 
−
- transfer 100 µl from every last well into the liquid waste<br/>
 
−
 
−
- repeat these steps for rpw B, C and D<br/>
 
−
 
−
- measure the fluorescence intensity in the plate reader<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit39"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">uM Fluorescein</th><th class="col1">50,00</th><th class="col2">25</th><th class="col3">12,5</th><th class="col4">6,25</th><th class="col5">3,125</th><th class="col6">1,5625</th><th class="col7">0,78125</th><th class="col8">0,390625</th><th class="col9">0,1953125</th><th class="col10">0,09765625</th><th class="col11">0,048828125</th><th class="col12">0</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">Replicate 1</td><td class="col1 rightalign"> 41550</td><td class="col2 rightalign"> 28587</td><td class="col3 rightalign"> 15976</td><td class="col4 rightalign"> 9537</td><td class="col5 rightalign"> 5674</td><td class="col6 rightalign"> 2712</td><td class="col7 rightalign"> 1357</td><td class="col8 rightalign"> 639</td><td class="col9 rightalign"> 348</td><td class="col10 rightalign"> 160</td><td class="col11 rightalign"> 91</td><td class="col12 rightalign"> 0</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">Replicate 2</td><td class="col1 rightalign"> 42434</td><td class="col2 rightalign"> 29060</td><td class="col3 rightalign"> 17592</td><td class="col4 rightalign"> 10169</td><td class="col5 rightalign"> 5250</td><td class="col6 rightalign"> 2705</td><td class="col7 rightalign"> 1155</td><td class="col8 rightalign"> 581</td><td class="col9 rightalign"> 302</td><td class="col10 rightalign"> 160</td><td class="col11 rightalign"> 67</td><td class="col12 rightalign"> 11</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">Replicate 3</td><td class="col1 rightalign"> 42066</td><td class="col2 rightalign"> 29363</td><td class="col3 rightalign"> 17743</td><td class="col4 rightalign"> 9882</td><td class="col5 rightalign"> 5015</td><td class="col6 rightalign"> 2671</td><td class="col7 rightalign"> 1303</td><td class="col8 rightalign"> 665</td><td class="col9 rightalign"> 314</td><td class="col10 rightalign"> 187</td><td class="col11 rightalign"> 87</td><td class="col12 rightalign"> 0</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">Replicate 4</td><td class="col1 rightalign"> 40538</td><td class="col2 rightalign"> 28766</td><td class="col3 rightalign"> 17808</td><td class="col4 rightalign"> 9934</td><td class="col5 rightalign"> 5127</td><td class="col6 rightalign"> 2818</td><td class="col7 rightalign"> 1344</td><td class="col8 rightalign"> 662</td><td class="col9 rightalign"> 366</td><td class="col10 rightalign"> 176</td><td class="col11 rightalign"> 113</td><td class="col12 rightalign"> 13</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_39'> </div>
 
−
<p>
 
−
<a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=picture1.png" class="media" title="picture1.png"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=8acd18&amp;media=picture1.png" class="media" alt="" width="400" /></a>
 
−
<a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=picture2.png" class="media" title="picture2.png"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=bfe4be&amp;media=picture2.png" class="media" alt="" width="400" /></a>
 
−
</p>
 
−
<img src="https://static.igem.org/mediawiki/2017/3/3a/T-Freiburg-8_5_2017_Interlab_Study_1.png">
 
−
<img src="https://static.igem.org/mediawiki/2017/e/e7/T-Freiburg-8_5_2017_Interlab_Study_2.png">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_38'> </div>
 
−
<h2 class="sectionedit40" id="section060817">06.08.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_40'> </div>
 
−
<h3 class="sectionedit41" id="pick_colonies_from_interlab_study">pick colonies from Interlab Study</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- pick 2 colonies from each plate<br/>
 
−
 
−
- incubation overnight at 37°C<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_41'> </div>
 
−
<h2 class="sectionedit42" id="section070817">07.08.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_42'> </div>
 
−
<h3 class="sectionedit43" id="interlab_study_cell_measurements">Interlab Study cell measurements</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- measure OD600 of the overnight cultures<br/>
 
−
 
−
- dilute the bacteria in medium with Chloramphenicol to a final OD = 0.02<br/>
 
−
 
−
- incubation at 37°C and 220 rpm<br/>
 
−
 
−
- take 500 µl of the cultures after 0h, 2h, 4h and 6h and place samples on ice<br/>
 
−
 
−
- transfer four replicates of 100 µl of each sample into a well of a 96-well-plate<br/>
 
−
 
−
- measure OD600 and fluorescence intensity in the plate reader (settings: excitation: 485 nm; emission: 530/30)<br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
<strong>Abs600 after 0h:</strong>
 
−
</p>
 
−
<div class="table sectionedit44"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0"> </th><th class="col1">Neg. Control</th><th class="col2 rightalign"> Pos. Control</th><th class="col3 rightalign"> Device 1</th><th class="col4 rightalign"> Device 2</th><th class="col5 rightalign"> Device 3</th><th class="col6 rightalign"> Device 4</th><th class="col7 rightalign"> Device 5</th><th class="col8 rightalign"> Device 6</th><th class="col9 rightalign"> LB + Chlor (blank)</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0 leftalign">Colony 1, Replicate 1 </td><td class="col1 leftalign">0,044 </td><td class="col2 leftalign">0,042 </td><td class="col3 leftalign">0,045 </td><td class="col4 leftalign">0,045 </td><td class="col5 leftalign">0,046 </td><td class="col6 leftalign">0,045 </td><td class="col7 leftalign">0,052 </td><td class="col8 leftalign">0,043 </td><td class="col9">0,041</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0 leftalign">Colony 1, Replicate 2 </td><td class="col1 leftalign">0,046 </td><td class="col2 leftalign">0,047 </td><td class="col3 leftalign">0,045 </td><td class="col4 leftalign">0,045 </td><td class="col5 leftalign">0,047 </td><td class="col6 leftalign">0,048 </td><td class="col7 leftalign">0,046 </td><td class="col8 leftalign">0,064 </td><td class="col9">0,041</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0 leftalign">Colony 1, Replicate 3 </td><td class="col1 leftalign">0,044 </td><td class="col2 leftalign">0,045 </td><td class="col3 leftalign">0,047 </td><td class="col4 leftalign">0,044 </td><td class="col5 leftalign">0,054 </td><td class="col6 leftalign">0,046 </td><td class="col7 leftalign">0,045 </td><td class="col8 leftalign">0,045 </td><td class="col9">0,043</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0 leftalign">Colony 1, Replicate 4 </td><td class="col1 leftalign">0,045 </td><td class="col2 leftalign">0,045 </td><td class="col3 leftalign">0,045 </td><td class="col4 leftalign">0,058 </td><td class="col5 leftalign">0,045 </td><td class="col6 leftalign">0,046 </td><td class="col7 leftalign">0,044 </td><td class="col8 leftalign">0,046 </td><td class="col9">0,046</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0 leftalign">Colony 2, Replicate 1 </td><td class="col1 leftalign">0,046 </td><td class="col2 leftalign">0,047 </td><td class="col3 leftalign">0,047 </td><td class="col4 leftalign">0,05 </td><td class="col5 leftalign">0,046 </td><td class="col6 leftalign">0,044 </td><td class="col7 leftalign">0,044 </td><td class="col8 leftalign">0,044 </td><td class="col9">0,043</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0 leftalign">Colony 2, Replicate 2 </td><td class="col1 leftalign">0,046 </td><td class="col2 leftalign">0,05 </td><td class="col3 leftalign">0,045 </td><td class="col4 leftalign">0,045 </td><td class="col5 leftalign">0,045 </td><td class="col6 leftalign">0,046 </td><td class="col7 leftalign">0,05 </td><td class="col8 leftalign">0,046 </td><td class="col9">0,046</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0 leftalign">Colony 2, Replicate 3 </td><td class="col1 leftalign">0,049 </td><td class="col2 leftalign">0,049 </td><td class="col3 leftalign">0,046 </td><td class="col4 leftalign">0,045 </td><td class="col5 leftalign">0,053 </td><td class="col6 leftalign">0,046 </td><td class="col7 leftalign">0,047 </td><td class="col8 leftalign">0,047 </td><td class="col9">0,044</td>
 
−
</tr>
 
−
<tr class="row8">
 
−
<td class="col0 leftalign">Colony 2, Replicate 4 </td><td class="col1 leftalign">0,046 </td><td class="col2 leftalign">0,051 </td><td class="col3 leftalign">0,043 </td><td class="col4 leftalign">0,043 </td><td class="col5 leftalign">0,044 </td><td class="col6 leftalign">0,049 </td><td class="col7 leftalign">0,048 </td><td class="col8 leftalign">0,05 </td><td class="col9">0,042</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_44'> </div>
 
−
<p>
 
−
<strong>Fluorescence intensity after 0h:</strong>
 
−
</p>
 
−
<div class="table sectionedit45"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0"> </th><th class="col1 rightalign"> Neg. Control</th><th class="col2 centeralign"> Pos. Control </th><th class="col3">Device 1</th><th class="col4 centeralign"> Device 2 </th><th class="col5">Device 3</th><th class="col6 centeralign"> Device 4 </th><th class="col7">Device 5</th><th class="col8 centeralign"> Device 6 </th><th class="col9">LB + Chlor (blank)</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0 leftalign">Colony 1, Replicate 1 </td><td class="col1 leftalign">30032 </td><td class="col2 leftalign">29468 </td><td class="col3 leftalign">47522 </td><td class="col4 leftalign">30425 </td><td class="col5 leftalign">37202 </td><td class="col6 leftalign">30859 </td><td class="col7 leftalign">38967 </td><td class="col8 leftalign">39848 </td><td class="col9">40167</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0 leftalign">Colony 1, Replicate 2 </td><td class="col1 leftalign">38825 </td><td class="col2 leftalign">32134 </td><td class="col3 leftalign">33823 </td><td class="col4 leftalign">37326 </td><td class="col5 leftalign">38129 </td><td class="col6 leftalign">41631 </td><td class="col7 leftalign">37103 </td><td class="col8 leftalign">40926 </td><td class="col9">383</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0 leftalign">Colony 1, Replicate 3 </td><td class="col1 leftalign">33426 </td><td class="col2 leftalign">38291 </td><td class="col3 leftalign">40791 </td><td class="col4 leftalign">38829 </td><td class="col5 leftalign">35103 </td><td class="col6 leftalign">35503 </td><td class="col7 leftalign">39082 </td><td class="col8 leftalign">38194 </td><td class="col9">40506</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0 leftalign">Colony 1, Replicate 4 </td><td class="col1 leftalign">30451 </td><td class="col2 leftalign">31664 </td><td class="col3 leftalign">40586 </td><td class="col4 leftalign">40661 </td><td class="col5 leftalign">37774 </td><td class="col6 leftalign">40046 </td><td class="col7 leftalign">38716 </td><td class="col8 leftalign">42854 </td><td class="col9">39830</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0 leftalign">Colony 2, Replicate 1 </td><td class="col1 leftalign">31429 </td><td class="col2 leftalign">32597 </td><td class="col3 leftalign">33648 </td><td class="col4 leftalign">34085 </td><td class="col5 leftalign">40700 </td><td class="col6 leftalign">31595 </td><td class="col7 leftalign">28914 </td><td class="col8 leftalign">36532 </td><td class="col9">40434</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0 leftalign">Colony 2, Replicate 2 </td><td class="col1 leftalign">39464 </td><td class="col2 leftalign">32361 </td><td class="col3 leftalign">39432 </td><td class="col4 leftalign">40150 </td><td class="col5 leftalign">39543 </td><td class="col6 leftalign">41282 </td><td class="col7 leftalign">38247 </td><td class="col8 leftalign">33320 </td><td class="col9">39231</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0 leftalign">Colony 2, Replicate 3 </td><td class="col1 leftalign">32329 </td><td class="col2 leftalign">31485 </td><td class="col3 leftalign">34366 </td><td class="col4 leftalign">36754 </td><td class="col5 leftalign">39980 </td><td class="col6 leftalign">41296 </td><td class="col7 leftalign">33348 </td><td class="col8 leftalign">33391 </td><td class="col9">39612</td>
 
−
</tr>
 
−
<tr class="row8">
 
−
<td class="col0 leftalign">Colony 2, Replicate 4 </td><td class="col1 leftalign">31225 </td><td class="col2 leftalign">36435 </td><td class="col3 leftalign">37063 </td><td class="col4 leftalign">38806 </td><td class="col5 leftalign">42787 </td><td class="col6 leftalign">40222 </td><td class="col7 leftalign">36968 </td><td class="col8 leftalign">36323 </td><td class="col9">38492</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_45'> </div>
 
−
<p>
 
−
<strong>Abs600 after 2h:</strong>
 
−
</p>
 
−
<div class="table sectionedit46"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0"> </th><th class="col1 leftalign">Neg. Control </th><th class="col2">Pos. Control</th><th class="col3 centeralign"> Device 1 </th><th class="col4">Device 2</th><th class="col5 centeralign"> Device 3 </th><th class="col6">Device 4</th><th class="col7 centeralign"> Device 5 </th><th class="col8">Device 6</th><th class="col9 rightalign"> LB + Chlor (blank)</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0 leftalign">Colony 1, Replicate 1 </td><td class="col1 leftalign">0,046 </td><td class="col2 leftalign">0,045 </td><td class="col3 leftalign">0,045 </td><td class="col4 leftalign">0,065 </td><td class="col5 leftalign">0,047 </td><td class="col6 leftalign">0,047 </td><td class="col7 leftalign">0,046 </td><td class="col8 leftalign">0,044 </td><td class="col9">0,041</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0 leftalign">Colony 1, Replicate 2 </td><td class="col1 leftalign">0,047 </td><td class="col2 leftalign">0,047 </td><td class="col3 leftalign">0,046 </td><td class="col4 leftalign">0,048 </td><td class="col5 leftalign">0,048 </td><td class="col6 leftalign">0,046 </td><td class="col7 leftalign">0,047 </td><td class="col8 leftalign">0,046 </td><td class="col9">0,043</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0 leftalign">Colony 1, Replicate 3 </td><td class="col1 leftalign">0,046 </td><td class="col2 leftalign">0,047 </td><td class="col3 leftalign">0,046 </td><td class="col4 leftalign">0,045 </td><td class="col5 leftalign">0,053 </td><td class="col6 leftalign">0,047 </td><td class="col7 leftalign">0,048 </td><td class="col8 leftalign">0,045 </td><td class="col9">0,044</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0 leftalign">Colony 1, Replicate 4 </td><td class="col1 leftalign">0,047 </td><td class="col2 leftalign">0,047 </td><td class="col3 leftalign">0,055 </td><td class="col4 leftalign">0,058 </td><td class="col5 leftalign">0,047 </td><td class="col6 leftalign">0,046 </td><td class="col7 leftalign">0,045 </td><td class="col8 leftalign">0,046 </td><td class="col9">0,043</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0 leftalign">Colony 2, Replicate 1 </td><td class="col1 leftalign">0,078 </td><td class="col2 leftalign">0,048 </td><td class="col3 leftalign">0,048 </td><td class="col4 leftalign">0,049 </td><td class="col5 leftalign">0,048 </td><td class="col6 leftalign">0,047 </td><td class="col7 leftalign">0,046 </td><td class="col8 leftalign">0,047 </td><td class="col9">0,043</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0 leftalign">Colony 2, Replicate 2 </td><td class="col1 leftalign">0,048 </td><td class="col2 leftalign">0,055 </td><td class="col3 leftalign">0,048 </td><td class="col4 leftalign">0,049 </td><td class="col5 leftalign">0,047 </td><td class="col6 leftalign">0,046 </td><td class="col7 leftalign">0,049 </td><td class="col8 leftalign">0,049 </td><td class="col9">0,041</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0 leftalign">Colony 2, Replicate 3 </td><td class="col1 leftalign">0,047 </td><td class="col2 leftalign">0,048 </td><td class="col3 leftalign">0,048 </td><td class="col4 leftalign">0,048 </td><td class="col5 leftalign">0,047 </td><td class="col6 leftalign">0,049 </td><td class="col7 leftalign">0,048 </td><td class="col8 leftalign">0,05 </td><td class="col9">0,045</td>
 
−
</tr>
 
−
<tr class="row8">
 
−
<td class="col0 leftalign">Colony 2, Replicate 4 </td><td class="col1 leftalign">0,046 </td><td class="col2 leftalign">0,049 </td><td class="col3 leftalign">0,047 </td><td class="col4 leftalign">0,046 </td><td class="col5 leftalign">0,045 </td><td class="col6 leftalign">0,048 </td><td class="col7 leftalign">0,048 </td><td class="col8 leftalign">0,049 </td><td class="col9">0,047</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_46'> </div>
 
−
<p>
 
−
<strong>Fluorescence intensity after 2h:</strong>
 
−
</p>
 
−
<div class="table sectionedit47"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0"> </th><th class="col1 centeralign"> Neg. Control </th><th class="col2">Pos. Control</th><th class="col3 centeralign"> Device 1 </th><th class="col4">Device 2</th><th class="col5 centeralign"> Device 3 </th><th class="col6">Device 4</th><th class="col7 rightalign"> Device 5</th><th class="col8 centeralign"> Device 6 </th><th class="col9">LB + Chlor (blank)</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0 leftalign">Colony 1, Replicate 1 </td><td class="col1 leftalign">32478 </td><td class="col2 leftalign">41305 </td><td class="col3 leftalign">33618 </td><td class="col4 leftalign">42435 </td><td class="col5 leftalign">34249 </td><td class="col6 leftalign">34205 </td><td class="col7 leftalign">34202 </td><td class="col8 leftalign">34949 </td><td class="col9">29069</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0 leftalign">Colony 1, Replicate 2 </td><td class="col1 leftalign">34345 </td><td class="col2 leftalign">42706 </td><td class="col3 leftalign">38570 </td><td class="col4 leftalign">43860 </td><td class="col5 leftalign">36547 </td><td class="col6 leftalign">44267 </td><td class="col7 leftalign">45267 </td><td class="col8 leftalign">47187 </td><td class="col9">37138</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0 leftalign">Colony 1, Replicate 3 </td><td class="col1 leftalign">38418 </td><td class="col2 leftalign">39069 </td><td class="col3 leftalign">38866 </td><td class="col4 leftalign">44974 </td><td class="col5 leftalign">36631 </td><td class="col6 leftalign">43986 </td><td class="col7 leftalign">46760 </td><td class="col8 leftalign">39103 </td><td class="col9">39152</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0 leftalign">Colony 1, Replicate 4 </td><td class="col1 leftalign">36379 </td><td class="col2 leftalign">42391 </td><td class="col3 leftalign">36129 </td><td class="col4 leftalign">45361 </td><td class="col5 leftalign">39003 </td><td class="col6 leftalign">44229 </td><td class="col7 leftalign">32123 </td><td class="col8 leftalign">33637 </td><td class="col9">41422</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0 leftalign">Colony 2, Replicate 1 </td><td class="col1 leftalign">36339 </td><td class="col2 leftalign">36658 </td><td class="col3 leftalign">36464 </td><td class="col4 leftalign">35639 </td><td class="col5 leftalign">43270 </td><td class="col6 leftalign">42121 </td><td class="col7 leftalign">34385 </td><td class="col8 leftalign">44627 </td><td class="col9">40854</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0 leftalign">Colony 2, Replicate 2 </td><td class="col1 leftalign">40618 </td><td class="col2 leftalign">43596 </td><td class="col3 leftalign">36428 </td><td class="col4 leftalign">44147 </td><td class="col5 leftalign">44768 </td><td class="col6 leftalign">43257 </td><td class="col7 leftalign">45151 </td><td class="col8 leftalign">46402 </td><td class="col9">39718</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0 leftalign">Colony 2, Replicate 3 </td><td class="col1 leftalign">39505 </td><td class="col2 leftalign">36517 </td><td class="col3 leftalign">43094 </td><td class="col4 leftalign">44747 </td><td class="col5 leftalign">36790 </td><td class="col6 leftalign">40034 </td><td class="col7 leftalign">44931 </td><td class="col8 leftalign">40597 </td><td class="col9">39712</td>
 
−
</tr>
 
−
<tr class="row8">
 
−
<td class="col0 leftalign">Colony 2, Replicate 4 </td><td class="col1 leftalign">36751 </td><td class="col2 leftalign">41857 </td><td class="col3 leftalign">35023 </td><td class="col4 leftalign">41187 </td><td class="col5 leftalign">35201 </td><td class="col6 leftalign">43423 </td><td class="col7 leftalign">32927 </td><td class="col8 leftalign">35063 </td><td class="col9">37786</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_47'> </div>
 
−
<p>
 
−
<strong>Abs600 after 4h:</strong>
 
−
</p>
 
−
<div class="table sectionedit48"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0"> </th><th class="col1 centeralign"> Neg. Control </th><th class="col2 leftalign">Pos. Control </th><th class="col3 leftalign">Device 1 </th><th class="col4">Device 2</th><th class="col5 centeralign"> Device 3 </th><th class="col6">Device 4</th><th class="col7 rightalign"> Device 5</th><th class="col8 rightalign"> Device 6</th><th class="col9 rightalign"> LB + Chlor (blank)</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0 leftalign">Colony 1, Replicate 1 </td><td class="col1 leftalign">0,056 </td><td class="col2 leftalign">0,055 </td><td class="col3 leftalign">0,055 </td><td class="col4 leftalign">0,052 </td><td class="col5 leftalign">0,058 </td><td class="col6 leftalign">0,052 </td><td class="col7 leftalign">0,053 </td><td class="col8 leftalign">0,046 </td><td class="col9">0,042</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0 leftalign">Colony 1, Replicate 2 </td><td class="col1 leftalign">0,054 </td><td class="col2 leftalign">0,056 </td><td class="col3 leftalign">0,054 </td><td class="col4 leftalign">0,052 </td><td class="col5 leftalign">0,055 </td><td class="col6 leftalign">0,054 </td><td class="col7 leftalign">0,055 </td><td class="col8 leftalign">0,048 </td><td class="col9">0,041</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0 leftalign">Colony 1, Replicate 3 </td><td class="col1 leftalign">0,055 </td><td class="col2 leftalign">0,061 </td><td class="col3 leftalign">0,055 </td><td class="col4 leftalign">0,052 </td><td class="col5 leftalign">0,057 </td><td class="col6 leftalign">0,052 </td><td class="col7 leftalign">0,055 </td><td class="col8 leftalign">0,049 </td><td class="col9">0,043</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0 leftalign">Colony 1, Replicate 4 </td><td class="col1 leftalign">0,055 </td><td class="col2 leftalign">0,056 </td><td class="col3 leftalign">0,05 </td><td class="col4 leftalign">0,053 </td><td class="col5 leftalign">0,053 </td><td class="col6 leftalign">0,052 </td><td class="col7 leftalign">0,05 </td><td class="col8 leftalign">0,049 </td><td class="col9">0,043</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0 leftalign">Colony 2, Replicate 1 </td><td class="col1 leftalign">0,053 </td><td class="col2 leftalign">0,059 </td><td class="col3 leftalign">0,057 </td><td class="col4 leftalign">0,061 </td><td class="col5 leftalign">0,053 </td><td class="col6 leftalign">0,059 </td><td class="col7 leftalign">0,053 </td><td class="col8 leftalign">0,059 </td><td class="col9">0,043</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0 leftalign">Colony 2, Replicate 2 </td><td class="col1 leftalign">0,054 </td><td class="col2 leftalign">0,056 </td><td class="col3 leftalign">0,058 </td><td class="col4 leftalign">0,062 </td><td class="col5 leftalign">0,053 </td><td class="col6 leftalign">0,059 </td><td class="col7 leftalign">0,055 </td><td class="col8 leftalign">0,059 </td><td class="col9">0,043</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0 leftalign">Colony 2, Replicate 3 </td><td class="col1 leftalign">0,054 </td><td class="col2 leftalign">0,056 </td><td class="col3 leftalign">0,058 </td><td class="col4 leftalign">0,063 </td><td class="col5 leftalign">0,057 </td><td class="col6 leftalign">0,059 </td><td class="col7 leftalign">0,055 </td><td class="col8 leftalign">0,06 </td><td class="col9">0,043</td>
 
−
</tr>
 
−
<tr class="row8">
 
−
<td class="col0 leftalign">Colony 2, Replicate 4 </td><td class="col1 leftalign">0,054 </td><td class="col2 leftalign">0,056 </td><td class="col3 leftalign">0,056 </td><td class="col4 leftalign">0,058 </td><td class="col5 leftalign">0,049 </td><td class="col6 leftalign">0,058 </td><td class="col7 leftalign">0,056 </td><td class="col8 leftalign">0,061 </td><td class="col9">0,042</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_48'> </div>
 
−
<p>
 
−
<strong>Fluorescence intensity after 4h:
 
−
</strong>
 
−
</p>
 
−
<div class="table sectionedit49"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0"> </th><th class="col1 centeralign"> Neg. Control </th><th class="col2">Pos. Control</th><th class="col3 centeralign"> Device 1 </th><th class="col4">Device 2</th><th class="col5 centeralign"> Device 3 </th><th class="col6">Device 4</th><th class="col7 rightalign"> Device 5</th><th class="col8 rightalign"> Device 6</th><th class="col9 rightalign"> LB + Chlor (blank)</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0 leftalign">Colony 1, Replicate 1 </td><td class="col1 leftalign">39398 </td><td class="col2 leftalign">41211 </td><td class="col3 leftalign">43472 </td><td class="col4 leftalign">34629 </td><td class="col5 leftalign">40024 </td><td class="col6 leftalign">37129 </td><td class="col7 leftalign">34265 </td><td class="col8 leftalign">40517 </td><td class="col9">38102</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0 leftalign">Colony 1, Replicate 2 </td><td class="col1 leftalign">39957 </td><td class="col2 leftalign">41499 </td><td class="col3 leftalign">44252 </td><td class="col4 leftalign">43313 </td><td class="col5 leftalign">44778 </td><td class="col6 leftalign">36906 </td><td class="col7 leftalign">43865 </td><td class="col8 leftalign">42985 </td><td class="col9">38644</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0 leftalign">Colony 1, Replicate 3 </td><td class="col1 leftalign">39198 </td><td class="col2 leftalign">41382 </td><td class="col3 leftalign">35548 </td><td class="col4 leftalign">45574 </td><td class="col5 leftalign">47239 </td><td class="col6 leftalign">39866 </td><td class="col7 leftalign">44132 </td><td class="col8 leftalign">44431 </td><td class="col9">37943</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0 leftalign">Colony 1, Replicate 4 </td><td class="col1 leftalign">35352 </td><td class="col2 leftalign">41590 </td><td class="col3 leftalign">26466 </td><td class="col4 leftalign">44768 </td><td class="col5 leftalign">45637 </td><td class="col6 leftalign">44736 </td><td class="col7 leftalign">29248 </td><td class="col8 leftalign">37825 </td><td class="col9">38618</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0 leftalign">Colony 2, Replicate 1 </td><td class="col1 leftalign">41468 </td><td class="col2 leftalign">42577 </td><td class="col3 leftalign">34717 </td><td class="col4 leftalign">39284 </td><td class="col5 leftalign">40334 </td><td class="col6 leftalign">42558 </td><td class="col7 leftalign">46026 </td><td class="col8 leftalign">41271 </td><td class="col9">39053</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0 leftalign">Colony 2, Replicate 2 </td><td class="col1 leftalign">43892 </td><td class="col2 leftalign">40143 </td><td class="col3 leftalign">36619 </td><td class="col4 leftalign">42028 </td><td class="col5 leftalign">41878 </td><td class="col6 leftalign">43940 </td><td class="col7 leftalign">38303 </td><td class="col8 leftalign">47567 </td><td class="col9">37978</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0 leftalign">Colony 2, Replicate 3 </td><td class="col1 leftalign">43598 </td><td class="col2 leftalign">43486 </td><td class="col3 leftalign">44578 </td><td class="col4 leftalign">44449 </td><td class="col5 leftalign">44828 </td><td class="col6 leftalign">44235 </td><td class="col7 leftalign">42442 </td><td class="col8 leftalign">37273 </td><td class="col9">39116</td>
 
−
</tr>
 
−
<tr class="row8">
 
−
<td class="col0 leftalign">Colony 2, Replicate 4 </td><td class="col1 leftalign">43048 </td><td class="col2 leftalign">43193 </td><td class="col3 leftalign">42421 </td><td class="col4 leftalign">38871 </td><td class="col5 leftalign">34908 </td><td class="col6 leftalign">41518 </td><td class="col7 leftalign">43876 </td><td class="col8 leftalign">44582 </td><td class="col9">37838</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_49'> </div>
 
−
<p>
 
−
<strong>Abs600 after 6h:</strong>
 
−
</p>
 
−
<div class="table sectionedit50"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0"> </th><th class="col1 leftalign">Neg. Control </th><th class="col2">Pos. Control</th><th class="col3 centeralign"> Device 1 </th><th class="col4">Device 2</th><th class="col5 rightalign"> Device 3</th><th class="col6 rightalign"> Device 4</th><th class="col7 rightalign"> Device 5</th><th class="col8 rightalign"> Device 6</th><th class="col9 rightalign"> LB + Chlor (blank)</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0 leftalign">Colony 1, Replicate 1 </td><td class="col1 leftalign">0,098 </td><td class="col2 leftalign">0,093 </td><td class="col3 leftalign">0,084 </td><td class="col4 leftalign">0,084 </td><td class="col5 leftalign">0,087 </td><td class="col6 leftalign">0,078 </td><td class="col7 leftalign">0,083 </td><td class="col8 leftalign">0,062 </td><td class="col9">0,048</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0 leftalign">Colony 1, Replicate 2 </td><td class="col1 leftalign">0,09 </td><td class="col2 leftalign">0,094 </td><td class="col3 leftalign">0,082 </td><td class="col4 leftalign">0,076 </td><td class="col5 leftalign">0,089 </td><td class="col6 leftalign">0,083 </td><td class="col7 leftalign">0,091 </td><td class="col8 leftalign">0,064 </td><td class="col9">0,041</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0 leftalign">Colony 1, Replicate 3 </td><td class="col1 leftalign">0,091 </td><td class="col2 leftalign">0,09 </td><td class="col3 leftalign">0,92 </td><td class="col4 leftalign">0,082 </td><td class="col5 leftalign">0,089 </td><td class="col6 leftalign">0,08 </td><td class="col7 leftalign">0,086 </td><td class="col8 leftalign">0,066 </td><td class="col9">0,043</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0 leftalign">Colony 1, Replicate 4 </td><td class="col1 leftalign">0,086 </td><td class="col2 leftalign">0,084 </td><td class="col3 leftalign">0,09 </td><td class="col4 leftalign">0,079 </td><td class="col5 leftalign">0,086 </td><td class="col6 leftalign">0,078 </td><td class="col7 leftalign">0,084 </td><td class="col8 leftalign">0,063 </td><td class="col9">0,043</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0 leftalign">Colony 2, Replicate 1 </td><td class="col1 leftalign">0,081 </td><td class="col2 leftalign">0,093 </td><td class="col3 leftalign">0,094 </td><td class="col4 leftalign">0,076 </td><td class="col5 leftalign">0,075 </td><td class="col6 leftalign">0,097 </td><td class="col7 leftalign">0,087 </td><td class="col8 leftalign">0,097 </td><td class="col9">0,043</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0 leftalign">Colony 2, Replicate 2 </td><td class="col1 leftalign">0,079 </td><td class="col2 leftalign">0,092 </td><td class="col3 leftalign">0,091 </td><td class="col4 leftalign">0,099 </td><td class="col5 leftalign">0,073 </td><td class="col6 leftalign">0,095 </td><td class="col7 leftalign">0,085 </td><td class="col8 leftalign">0,107 </td><td class="col9">0,043</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0 leftalign">Colony 2, Replicate 3 </td><td class="col1 leftalign">0,08 </td><td class="col2 leftalign">0,086 </td><td class="col3 leftalign">0,101 </td><td class="col4 leftalign">0,13 </td><td class="col5 leftalign">0,073 </td><td class="col6 leftalign">0,1 </td><td class="col7 leftalign">0,088 </td><td class="col8 leftalign">0,107 </td><td class="col9">0,042</td>
 
−
</tr>
 
−
<tr class="row8">
 
−
<td class="col0 leftalign">Colony 2, Replicate 4 </td><td class="col1 leftalign">0,078 </td><td class="col2 leftalign">0,092 </td><td class="col3 leftalign">0,089 </td><td class="col4 leftalign">0,108 </td><td class="col5 leftalign">0,073 </td><td class="col6 leftalign">0,093 </td><td class="col7 leftalign">0,09 </td><td class="col8 leftalign">0,105 </td><td class="col9">0,042</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_50'> </div>
 
−
<p>
 
−
<strong>Fluorescence intensity after 6h:</strong>
 
−
</p>
 
−
<div class="table sectionedit51"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0"> </th><th class="col1 rightalign"> Neg. Control</th><th class="col2 centeralign"> Pos. Control </th><th class="col3">Device 1</th><th class="col4 centeralign"> Device 2 </th><th class="col5">Device 3</th><th class="col6 rightalign"> Device 4</th><th class="col7 rightalign"> Device 5</th><th class="col8 centeralign"> Device 6 </th><th class="col9">LB + Chlor (blank)</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0 leftalign">Colony 1, Replicate 1 </td><td class="col1 leftalign">44819 </td><td class="col2 leftalign">43197 </td><td class="col3 leftalign">34990 </td><td class="col4 leftalign">39952 </td><td class="col5 leftalign">35312 </td><td class="col6 leftalign">44026 </td><td class="col7 leftalign">44108 </td><td class="col8 leftalign">34395 </td><td class="col9">41891</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0 leftalign">Colony 1, Replicate 2 </td><td class="col1 leftalign">35745 </td><td class="col2 leftalign">41624 </td><td class="col3 leftalign">42712 </td><td class="col4 leftalign">36773 </td><td class="col5 leftalign">38191 </td><td class="col6 leftalign">39327 </td><td class="col7 leftalign">36222 </td><td class="col8 leftalign">38684 </td><td class="col9">44486</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0 leftalign">Colony 1, Replicate 3 </td><td class="col1 leftalign">43503 </td><td class="col2 leftalign">43470 </td><td class="col3 leftalign">38934 </td><td class="col4 leftalign">39338 </td><td class="col5 leftalign">39338 </td><td class="col6 leftalign">47418 </td><td class="col7 leftalign">46563 </td><td class="col8 leftalign">41520 </td><td class="col9">40216</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0 leftalign">Colony 1, Replicate 4 </td><td class="col1 leftalign">40336 </td><td class="col2 leftalign">39507 </td><td class="col3 leftalign">37827 </td><td class="col4 leftalign">41517 </td><td class="col5 leftalign">41517 </td><td class="col6 leftalign">46271 </td><td class="col7 leftalign">47662 </td><td class="col8 leftalign">42298 </td><td class="col9">38353</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0 leftalign">Colony 2, Replicate 1 </td><td class="col1 leftalign">34982 </td><td class="col2 leftalign">34672 </td><td class="col3 leftalign">46394 </td><td class="col4 leftalign">18665 </td><td class="col5 leftalign">18665 </td><td class="col6 leftalign">36165 </td><td class="col7 leftalign">36374 </td><td class="col8 leftalign">40293 </td><td class="col9">40211</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0 leftalign">Colony 2, Replicate 2 </td><td class="col1 leftalign">43791 </td><td class="col2 leftalign">37273 </td><td class="col3 leftalign">44467 </td><td class="col4 leftalign">41079 </td><td class="col5 leftalign">41079 </td><td class="col6 leftalign">41516 </td><td class="col7 leftalign">44327 </td><td class="col8 leftalign">38827 </td><td class="col9">38014</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0 leftalign">Colony 2, Replicate 3 </td><td class="col1 leftalign">39248 </td><td class="col2 leftalign">40869 </td><td class="col3 leftalign">38169 </td><td class="col4 leftalign">48933 </td><td class="col5 leftalign">48933 </td><td class="col6 leftalign">44903 </td><td class="col7 leftalign">41210 </td><td class="col8 leftalign">38643 </td><td class="col9">40437</td>
 
−
</tr>
 
−
<tr class="row8">
 
−
<td class="col0 leftalign">Colony 2, Replicate 4 </td><td class="col1 leftalign">38078 </td><td class="col2 leftalign">38846 </td><td class="col3 leftalign">41889 </td><td class="col4 leftalign">49892 </td><td class="col5 leftalign">49892 </td><td class="col6 leftalign">42376 </td><td class="col7 leftalign">46132 </td><td class="col8 leftalign">38525 </td><td class="col9">39913</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_51'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_43'> </div>
 
−
<h2 class="sectionedit52" id="section1907270717">19.07.-27.07.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_52'> </div>
 
−
<h3 class="sectionedit53" id="ph_change_over_time_in_incubator_with_rpmi1640_and_acid">pH change over time in incubator with RPMI1640 and acid</h3>
 
−
<div class="level3">
 
−
<div class="table sectionedit54"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">composition</th><th class="col1">start pH</th><th class="col2">incubation time</th><th class="col3">trend</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2ml RPMI1640</td><td class="col1">7.14</td><td class="col2">93 min</td><td class="col3"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=no_acid_1.png" class="media" title="no_acid_1.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=2c37ff&amp;media=no_acid_1.png" class="mediacenter" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">2ml RPMI1640</td><td class="col1">7.16</td><td class="col2">67 min</td><td class="col3"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=no_acid_2.png" class="media" title="no_acid_2.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=794dc5&amp;media=no_acid_2.png" class="mediacenter" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">2ml RPMI1640</td><td class="col1">7.15</td><td class="col2">104 min</td><td class="col3"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=no_acid_3.png" class="media" title="no_acid_3.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=2dd7e8&amp;media=no_acid_3.png" class="mediacenter" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">2ml RPMI1640 + 65µl lactic acid (0.6 M)</td><td class="col1">6.2</td><td class="col2">95 min</td><td class="col3"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=07-20-17_lactic_acid_no_cells.png" class="media" title="07-20-17_lactic_acid_no_cells.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=8aeac1&amp;media=07-20-17_lactic_acid_no_cells.png" class="mediacenter" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">2ml RPMI1649 + 65µl lactic acid (0.6 M)</td><td class="col1">6.3</td><td class="col2">90 min</td><td class="col3"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=07-21-17_lactic_acid_no_cells.png" class="media" title="07-21-17_lactic_acid_no_cells.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=2ac7e7&amp;media=07-21-17_lactic_acid_no_cells.png" class="mediacenter" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">2ml RPMI1640 + 20µl HCl(1.2 M)</td><td class="col1">6.6</td><td class="col2">105 min</td><td class="col3"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=07-20-17_hcl_no_cells.png" class="media" title="07-20-17_hcl_no_cells.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=3463ad&amp;media=07-20-17_hcl_no_cells.png" class="mediacenter" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0">2ml RPMI1640 + 30µl HCl(1.2 M)</td><td class="col1">6.6</td><td class="col2">100 min</td><td class="col3"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=07-21-17_hcl_no_cells.png" class="media" title="07-21-17_hcl_no_cells.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=0e0744&amp;media=07-21-17_hcl_no_cells.png" class="mediacenter" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row8">
 
−
<td class="col0">7.5 ml RPMI1640 + 250.000 HEK cells / ml</td><td class="col1">7.3</td><td class="col2">24 h</td><td class="col3"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=250k_cells_no_acid.png" class="media" title="250k_cells_no_acid.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=9a20f6&amp;media=250k_cells_no_acid.png" class="mediacenter" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row9">
 
−
<td class="col0">7.5 ml RPMI1640 + 500.000 HEK cells / ml</td><td class="col1">7.17</td><td class="col2">26 h</td><td class="col3"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=ph-test_19-08-17.jpg" class="media" title="ph-test_19-08-17.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=0735d0&amp;media=ph-test_19-08-17.jpg" class="mediacenter" alt="" width="200" /></a></td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_54'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_53'> </div>
 
−
<h2 class="sectionedit55" id="section100817">10.08.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_55'> </div>
 
−
<h3 class="sectionedit56" id="transformation_of_pwhe-644">Transformation of pWHE-644</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- transformation in DH5-α
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_56'> </div>
 
−
<h2 class="sectionedit57" id="section120817">12.08.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_57'> </div>
 
−
<h3 class="sectionedit58" id="miniprep_of_pwhe-644_in_dh3-α">Miniprep of pWHE-644 in DH3-α</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- miniprep with Zymo kit
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_58'> </div>
 
−
<h3 class="sectionedit59" id="test_digest_of_pwhe-644">Test digest of pWHE-644</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
1µl NcoI<br/>
 
−
 
−
2µl DNA<br/>
 
−
 
−
1.5µl FD-buffer 10x<br/>
 
−
 
−
10.5µl H20<br/>
 
−
 
−
Excepted bands could be observed.<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_59'> </div>
 
−
<h2 class="sectionedit60" id="section130817">13.08.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_60'> </div>
 
−
<h3 class="sectionedit61" id="miniprep_of_cmv-vegfr2_pig_138">Miniprep of CMV-VEGFR2 (pIG_138)</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- 7x miniprep of CMV-VEGFR2
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_61'> </div>
 
−
<h3 class="sectionedit62" id="test_digest_of_cmv-vegfr2_pig_138">Test digest of CMV-VEGFR2 (pIG_138)</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
2µl DNA<br/>
 
−
 
−
1.5µl buffer<br/>
 
−
 
−
1µl BamHI<br/>
 
−
 
−
0.5µl H20<br/>
 
−
 
−
Only one of the expected bands could be oberserved.<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_62'> </div>
 
−
<h2 class="sectionedit63" id="section1308171">13.08.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_63'> </div>
 
−
<h3 class="sectionedit64" id="test_digest_of_cmv-vegfr2_pig_1381">Test digest of CMV-VEGFR2 (pIG_138)</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
2µl DNA<br/>
 
−
 
−
1.5µl FD-buffer<br/>
 
−
 
−
1µl SmaI<br/>
 
−
 
−
Only one of the expected bands could be oberserved.<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_64'> </div>
 
−
<h2 class="sectionedit65" id="section160817">16.08.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_65'> </div>
 
−
<h3 class="sectionedit66" id="ph_test_with_jurkat_cells">pH test with Jurkat cells</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- centrifuge Jurkatt cells, dilute in RPMI1640<br/>
 
−
 
−
- count cells<br/>
 
−
 
−
- incubation of Jurkat cells and lactic acid<br/>
 
−
 
−
- pH measurements after 0h, 30min, 1h and 1h30min<br/>
 
−
 
−
- 1) only RPMI1640 with cells, start pH at 7.2<br/>
 
−
 
−
- 2) RPMI1640 with cells and 100µl lactic acid, start pH at 6.8<br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
after 0h:<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit67"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">cell concentration (cells/ml)</th><th class="col1">pH of 1)</th><th class="col2">pH of 2)</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">3mio</td><td class="col1">7.46</td><td class="col2">6.81</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">2mio</td><td class="col1">7.41</td><td class="col2">6.82</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">1.5mio</td><td class="col1">7.47</td><td class="col2">6.86</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">1mio</td><td class="col1">7.49</td><td class="col2">6.79</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">500k</td><td class="col1">7.45</td><td class="col2">6.78</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">100k</td><td class="col1">7.45</td><td class="col2">6.77</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_67'> </div>
 
−
<p>
 
−
after 30min:<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit68"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">cell concentration (cells/ml)</th><th class="col1">pH of 1)</th><th class="col2">pH of 2)</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">3mio</td><td class="col1">7.52</td><td class="col2">7.18</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">2mio</td><td class="col1">7.51</td><td class="col2">7.13</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">1.5mio</td><td class="col1">7.53</td><td class="col2">7.14</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">1mio</td><td class="col1">7.52</td><td class="col2">7.11</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">500k</td><td class="col1">7.54</td><td class="col2">7.07</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">100k</td><td class="col1">7.55</td><td class="col2">7.12</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_68'> </div>
 
−
<p>
 
−
after 1h:<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit69"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">cell concentration (cells/ml)</th><th class="col1">pH of 1)</th><th class="col2">pH of 2)</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">3mio</td><td class="col1">7.48</td><td class="col2">7.26</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">2mio</td><td class="col1">7.57</td><td class="col2">7.22</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">1.5mio</td><td class="col1">7.60</td><td class="col2">7.24</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">1mio</td><td class="col1">7.60</td><td class="col2">7.22</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">500k</td><td class="col1">7.60</td><td class="col2">7.18</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">100k</td><td class="col1">7.63</td><td class="col2">7.21</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_69'> </div>
 
−
<p>
 
−
after 1h30min:<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit70"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">cell concentration (cells/ml)</th><th class="col1">pH of 1)</th><th class="col2">pH of 2)</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">3mio</td><td class="col1">7.42</td><td class="col2">7.14</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">2mio</td><td class="col1">7.55</td><td class="col2">7.27</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">1.5mio</td><td class="col1">7.46</td><td class="col2">7.35</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">1mio</td><td class="col1">7.62</td><td class="col2">7.18</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">500k</td><td class="col1">7.51</td><td class="col2">7.23</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">100k</td><td class="col1">7.59</td><td class="col2">7.26</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_70'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_66'> </div>
 
−
<h2 class="sectionedit71" id="section1608171">16.08.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_71'> </div>
 
−
<h3 class="sectionedit72" id="test_digest_of_cmv-vegfr2_pig_1382">Test digest of CMV-VEGFR2 (pIG_138)</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
2µl DNA<br/>
 
−
 
−
1.5µl FD-buffer<br/>
 
−
 
−
2µl BamHI<br/>
 
−
 
−
9,5µl H20<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_72'> </div>
 
−
<h3 class="sectionedit73" id="ctla4_induction">Ctla4 induction</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- Jurkat cells transiently transfected with Ctla4-CFP-CMV-mCherry<br/>
 
−
 
−
- positive control: cotransfected with CMV-CFP and CMV mCherry<br/>
 
−
 
−
- add VEGF<br/>
 
−
 
−
- incubation for 24h at 37°C<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_73'> </div>
 
−
<h2 class="sectionedit74" id="section170817">17.08.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_74'> </div>
 
−
<h3 class="sectionedit75" id="pei_transfection_of_hek_cells">PEI transfection of HEK cells</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- CMV-CFP from iGEM<br/>
 
−
 
−
- CMV-CFP from tool box<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_75'> </div>
 
−
<h3 class="sectionedit76" id="flow_cytometry">Flow cytometry</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- Ctla4-CFP-CMV-mCherry
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_76'> </div>
 
−
<h2 class="sectionedit77" id="section180817">18.08.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_77'> </div>
 
−
<h3 class="sectionedit78" id="biorad_electroporation">BioRad electroporation</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Cell lines: Jurkat<br/>
 
−
 
−
Plasmids: pIG_037, pIG_031, contransfection CMV-CFP<br/>
 
−
 
−
</p>
 
−
<ol>
 
−
<li class="level1"><div class="li"> Count the cells</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Transfer 1 mio. cells into falcons and centrifuge with 100g for 3 min</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Resuspend the cell pellet with 100 µl P3 Buffer</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Add 9 µg pIG17_009</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Transfer the mixture into cuvette </div>
 
−
</li>
 
−
<li class="level1"><div class="li"> BioRad setting: 250 V, 960 µF, 35 sec.</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Add 500 µl RPMI medium to the cells immediately after electroporation</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Plate the cells into 12-well plate with 2 ml Medium</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Over night culturing</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Wash the cuvettes for reusing</div>
 
−
</li>
 
−
</ol>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_78'> </div>
 
−
<h3 class="sectionedit79" id="calcium_test">Calcium test</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- incubation of cells at 37°C in FACS tubes for 5min
 
−
- pre-FACS after 3min
 
−
- add VEGF (30ng/ml, 70ng/ml)
 
−
- FACS afer 7min: no signal
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_79'> </div>
 
−
<h2 class="sectionedit80" id="section200817">20.08.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_80'> </div>
 
−
<h3 class="sectionedit81" id="biorad_electroporation1">Biorad electroporation</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Cell lines: Jurkat<br/>
 
−
 
−
Plasmids: pIG_034, cotransfection CMV-CFP and CMV-mCherry, negative control (no DNA)<br/>
 
−
 
−
</p>
 
−
<ol>
 
−
<li class="level1"><div class="li"> Count the cells<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Transfer 1 mio. cells into falcons and centrifuge with 100g for 3 min<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Resuspend the cell pellet with 100 µl P3 Buffer<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Add 6 µg pIG17_009<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Transfer the mixture into cuvette <br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> BioRad setting: 250 V, 960 µF, 35 sec.<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Add 500 µl RPMI medium to the cells immediately after electroporation<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Plate the cells into 12-well plate with 2 ml Medium<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Over night culturing<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Wash the cuvettes for reusing<br/>
 
−
</div>
 
−
</li>
 
−
</ol>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_81'> </div>
 
−
<h3 class="sectionedit82" id="ph_tests_with_jurkat">pH tests with Jurkat</h3>
 
−
<div class="level3">
 
−
<div class="table sectionedit83"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">V(ges) [ml]</th><th class="col1">V(RPMI1640) [ml]</th><th class="col2">V(cells) [ml]</th><th class="col3">c(cells) [cells/ml]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">7.5</td><td class="col1">5.3</td><td class="col2">2.2</td><td class="col3">250.000</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_83'> </div>
 
−
<p>
 
−
- incubation for 24h
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_82'> </div>
 
−
<h3 class="sectionedit84" id="miniprep_of_pig_034">Miniprep of pIG_034</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- miniprep 8x
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_84'> </div>
 
−
<h2 class="sectionedit85" id="section210817">21.08.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_85'> </div>
 
−
<h3 class="sectionedit86" id="sequencing_of_pig_138">sequencing of pIG_138</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- plasmid: CMV-VEGFR2 (pIG_138)<br/>
 
−
 
−
- DNA concentration: 80ng/ml<br/>
 
−
 
−
- Oligo: 2µl DNA, 18µl H2O<br/>
 
−
 
−
- V(ges): 20µl<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_86'> </div>
 
−
<h2 class="sectionedit87" id="section220817">22.08.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_87'> </div>
 
−
<h3 class="sectionedit88" id="miniprep_of_pig_017_pig_031_pig_034_pig_037">Miniprep of pIG_017, pIG_031, pIG_034, pIG_037</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- Zymo kit<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_88'> </div>
 
−
<h3 class="sectionedit89" id="up-concentration_of_pig_017_pig_031_pig_034_pig_037">up-concentration of pIG_017, pIG_031, pIG_034, pIG_037</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- add isopropanol to plasmid suspension in H2O<br/>
 
−
 
−
- -20°C for 20min<br/>
 
−
 
−
- centrifugate for 30min at maximal speed and 4°C<br/>
 
−
 
−
- remove isopropanol<br/>
 
−
 
−
- wash with EtOH (70%)<br/>
 
−
 
−
- centrifugate for 30min att maximal speed and 4°C<br/>
 
−
 
−
- remove EtOH<br/>
 
−
 
−
- open eppis to dry overnight<br/>
 
−
 
−
- eluate with H2O<br/>
 
−
 
−
Up-conentration did not work, DNA concentration decreased.<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_89'> </div>
 
−
<h2 class="sectionedit90" id="section250817">25.08.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_90'> </div>
 
−
<h3 class="sectionedit91" id="miniprep_of_pig_017_pig_037">Miniprep of pIG_017, pIG_037</h3>
 
−
<div class="level3">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_91'> </div>
 
−
<h2 class="sectionedit92" id="section070917">07.09.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_92'> </div>
 
−
<h3 class="sectionedit93" id="pei_transfection_of_hek_cells1">PEI transfection of HEK cells</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
used cells: HEK cells<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit94"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">Plasmid number</th><th class="col1">component</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">pIG17_034</td><td class="col1">pCRE-CFP-CMV-mCherry</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pIG17_008 + pIG17_009</td><td class="col1">CMV-CFP + CMV-mCherry</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">pIG17_034 + β2-receptor</td><td class="col1">pCRE-CFP-CMV-mCherry + β2-receptor</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">no DNA</td><td class="col1">negative control</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_94'> </div><ol>
 
−
<li class="level1"><div class="li"> Split the cells 1:5 into 10 cm plate<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Thaw PEI reagent at RT<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> incubate 15 min at RT<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Add the mix to the culture<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> incubate for 24 h<br/>
 
−
</div>
 
−
</li>
 
−
</ol>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_93'> </div>
 
−
<h3 class="sectionedit95" id="gel_extraction_of_m1-receptor_without_cfp">Gel extraction of M1-receptor without CFP</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
50 ng DNA<br/>
 
−
 
−
1 µl T4 Ligase<br/>
 
−
 
−
2 µl Buffer 10x<br/>
 
−
 
−
13 µl H2O<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_95'> </div>
 
−
<h2 class="sectionedit96" id="section080917">08.09.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_96'> </div>
 
−
<h3 class="sectionedit97" id="colony_pcr_of_m1">Colony PCR of M1</h3>
 
−
<div class="level3">
 
−
<div class="table sectionedit98"><table class="inline">
 
−
<tr class="row0">
 
−
<td class="col0">Buffer 10x</td><td class="col1">2.5µl</td><td class="col2">12.5 µl</td>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">dNTP 10 mM</td><td class="col1">0.5 µl</td><td class="col2">2.5 µl</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">Template</td><td class="col1">1 µl</td><td class="col2"> </td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">Primer</td><td class="col1">0.5 µl</td><td class="col2">2.5 µl</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">DreamTaq Thermo Fisher</td><td class="col1">0.25 µl</td><td class="col2">1.25 µl</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">H2O</td><td class="col1">20 µl</td><td class="col2">100 µl</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_98'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_97'> </div>
 
−
<h2 class="sectionedit99" id="section2008171">20.08.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_99'> </div>
 
−
<h3 class="sectionedit100" id="pcre_induction">pCRE induction</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- treatment in 6-well-plate<br/>
 
−
 
−
- add Forskolin and β2-inductor to the cells<br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
<strong>Cells transfected with pIG17_034 (pCRE-CFP-CMV-mCherry):</strong>
 
−
</p>
 
−
<div class="table sectionedit101"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">approach</th><th class="col1">Forskolin</th><th class="col2">β2-inductor</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">negative control</td><td class="col1">-</td><td class="col2">-</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">old Forskolin</td><td class="col1">10 µM (old)</td><td class="col2">-</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">new Forskolin</td><td class="col1">10 µM (new)</td><td class="col2">-</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">new Forskolin</td><td class="col1">100 µM new Forskolin</td><td class="col2"></td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">β2-inductor</td><td class="col1">-</td><td class="col2">10 µM</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">β2-inductor</td><td class="col1">-</td><td class="col2">100 µM</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_101'> </div>
 
−
<p>
 
−
<strong>Cells transfected with pIG17_034 (pCRE-CFP-CMV-mCherry) and β2-receptor:
 
−
</strong>
 
−
</p>
 
−
<div class="table sectionedit102"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">approach</th><th class="col1">Forskolin</th><th class="col2">β2-inductor</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">negative control</td><td class="col1">-</td><td class="col2">-</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">β2-inductor</td><td class="col1">-</td><td class="col2">10 µM</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">β2-inductor</td><td class="col1">-</td><td class="col2">100 µM</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_102'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_100'> </div>
 
−
<h2 class="sectionedit103" id="section100917">10.09.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_103'> </div>
 
−
<h3 class="sectionedit104" id="pei_transfection_of_plasmids_pig17_022_23_37_861">PEI transfection of plasmids pIG17_022/23/37/86</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
used cells: HEK cells<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit105"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">Plasmid number</th><th class="col1">component</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">pIG17_031</td><td class="col1">HRE-CFP-CMV-mCherry</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_105'> </div><ol>
 
−
<li class="level1"><div class="li"> Split the cells 1:2 into 10 cm plate<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Thaw PEI reagent at RT<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> incubate 15 min at RT<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Add the mix to the culture<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> incubate for 24 h<br/>
 
−
</div>
 
−
</li>
 
−
</ol>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_104'> </div>
 
−
<h3 class="sectionedit106" id="hre_induction">HRE induction</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- add CoCl2 (end concentrations: 0 µM, 5 µM, 10 µM, 20 µM, 50 µM, 100 µM, 200 µM, 400 µM, 800 µM)<br/>
 
−
 
−
- fluoresence microscopy<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_106'> </div>
 
−
<h3 class="sectionedit107" id="repetition_of_the_interlab_study">Repetition of the Interlab Study</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- transformation of DH5-α with plasmids from Kit 7 (21B, 21D, 21F, 21H, 21L, 21N, 21P)<br/>
 
−
 
−
- incubation at 37 °C<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_107'> </div>
 
−
<h3 class="sectionedit108" id="pei_transfection_of_hek_cells2">PEI transfection of HEK cells</h3>
 
−
<div class="level3">
 
−
<div class="table sectionedit109"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">Plasmid number</th><th class="col1">component</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">pIG17_034 + L3 (Luciferase)</td><td class="col1">pCRE-CFP-CMV-mCherry + L3</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pIG17_034 + TDAG8</td><td class="col1">pCRE-CFP-CMV-mCherry + TDAG8</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">pIG17_008 + pIG17_009</td><td class="col1">CMV-CFP + CMV-mCherry</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_109'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_108'> </div>
 
−
<h2 class="sectionedit110" id="section160917">16.09.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_110'> </div>
 
−
<h3 class="sectionedit111" id="pick_colonies_of_interlab_study">Pick colonies of Interlab Study</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- 2 colonies per plate<br/>
 
−
 
−
- incubation at 37°C<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_111'> </div>
 
−
<h3 class="sectionedit112" id="pcre_induction_in_hek_cells">pCRE induction in HEK cells</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- plate out cells on 24-well-dish<br/>
 
−
 
−
- treatment with Forskolin<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit113"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">Plasmid number</th><th class="col1">component</th><th class="col2">Forskolin treatment</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">pIG17_008 + pIG17_009</td><td class="col1">CMV-CFP + CMV-mCherry</td><td class="col2">-</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pIG17_034 + TDAG8</td><td class="col1">pCRE-CFP-CMV-mCherry + TDAG8</td><td class="col2">+</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">no DNA</td><td class="col1">-</td><td class="col2">-</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">no DNA</td><td class="col1">-</td><td class="col2">+</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pIG17_034</td><td class="col1">pCRE-CFP-CMV-mCherry</td><td class="col2">-</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">pIG17_034</td><td class="col1">pCRE-CFP-CMV-mCherry</td><td class="col2">+</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_113'> </div>
 
−
<p>
 
−
- after 10 hours: pH change to 6.5, 7.0, 7.5<br/>
 
−
 
−
- plate reader after 1h, 3h, 6h, 12h, 24h<br/>
 
−
 
−
- FACS analysis after 24h<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_112'> </div>
 
−
<h2 class="sectionedit114" id="section170917">17.09.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_114'> </div>
 
−
<h3 class="sectionedit115" id="pei_transfection_of_plasmid_pig17_031">PEI transfection of plasmid pIG17_031</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
used cells: HEK cells<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit116"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">Plasmid number</th><th class="col1">component</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">pIG17_031</td><td class="col1">HRE-CFP-CMV-mCherry</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pIG17_008 + pIG17_009</td><td class="col1">CMV-CFP + CMV-mCherry</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_116'> </div><ol>
 
−
<li class="level1"><div class="li"> Split the cells 1:2 into 10 cm plate (on 16.09.17)<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Thaw PEI reagent at RT<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> incubate 15 min at RT<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Add the mix to the culture<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> incubate for 24 h<br/>
 
−
</div>
 
−
</li>
 
−
</ol>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_115'> </div>
 
−
<h2 class="sectionedit117" id="section180917">18.09.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_117'> </div>
 
−
<h3 class="sectionedit118" id="hre_induction1">HRE induction</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- induction cancelled because cells were contaminated with bacteria<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_118'> </div>
 
−
<h2 class="sectionedit119" id="section190917">19.09.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_119'> </div>
 
−
<h3 class="sectionedit120" id="overnights_of_overnights_hre4x_cre4x">overnights of overnights (HRE4x, CRE4x)</h3>
 
−
<div class="level3">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_120'> </div>
 
−
<h2 class="sectionedit121" id="section200917">20.09.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_121'> </div>
 
−
<h3 class="sectionedit122" id="midiprep_of_cultures_from_overnights">Midiprep of cultures from overnights</h3>
 
−
<div class="level3">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_122'> </div>
 
−
<h3 class="sectionedit123" id="pei_transfection">PEI transfection</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
used cells: HEK cells<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit124"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">Plasmid number</th><th class="col1">component</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0 rightalign">  + TDAG8</td><td class="col1">CRE(4x)-CFP-CMV-mCherry + TDAG8</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0"> </td><td class="col1">HRE(4x)-CFP-CMV-mCherry</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">pIG17_008 + pIF17_009</td><td class="col1">CMV-CFP + CMV-mCherry</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_124'> </div><ol>
 
−
<li class="level1"><div class="li"> Split the cells 1:2, transfer into 6-well-plate (on 16.09.17)<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Thaw PEI reagent at RT<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> incubate 15 min at RT<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Add the mix to the culture<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> incubate for 24 h<br/>
 
−
</div>
 
−
</li>
 
−
</ol>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_123'> </div>
 
−
<h3 class="sectionedit125" id="filtration_of_dmem_addition_of_fbs">Filtration of DMEM, addition of FBS</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- filter DMEM sterile<br/>
 
−
 
−
- add 2 % FBS<br/>
 
−
 
−
- set up pH of aliquots with HCl (4 M)<br/>
 
−
 
−
- final pH values: 6.7, 7.1, 7.4<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_125'> </div>
 
−
<h2 class="sectionedit126" id="section220917">22.09.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_126'> </div>
 
−
<h3 class="sectionedit127" id="induction_of_cre_and_hre_in_hek_cells">induction of CRE and HRE in HEK cells</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- indution in 96-wel-plates
 
−
- cells transiently transfected with CRE-CFP-CMV-mCherry, add acid<br/>
 
−
 
−
- cells transiently transfected with HRE-CFP-CMV-mCherry, add CoCl2<br/>
 
−
 
−
- plate reader: no measurable induction of CFP<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_127'> </div>
 
−
<h2 class="sectionedit128" id="section230917">23.09.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_128'> </div>
 
−
<h3 class="sectionedit129" id="facs_analysis_of_hek_cells">FACS analysis of HEK cells</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- FACS analysis after CRE induction and HRE induction<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_129'> </div>
 
−
<h2 class="sectionedit130" id="section240917">24.09.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_130'> </div>
 
−
<h3 class="sectionedit131" id="repetition_of_the_interlab_study1">Repetition of the Interlab Study</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- transformation of DH5-α with plasmids from Kit 6 (20B, 20D, 20F, 20H, 20L, 20N, 20P)<br/>
 
−
 
−
- incubation at 37 °C<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_131'> </div>
 
−
<h2 class="sectionedit132" id="section290917">29.09.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_132'> </div>
 
−
<h3 class="sectionedit133" id="calibration_of_plate_reader">calibration of plate reader</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- used cells: knockdown-cells + untransfected cells, compositions 1:0, 1:1, 1:2, 1:4, 0:1<br/>
 
−
 
−
- 96-well-plates, black, clear bottom, 100 000 cells per well<br/>
 
−
 
−
- different media: RPMI164, DMEM, DMEM and PBS (500 000 cells per well)
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_133'> </div>
 
−
<h3 class="sectionedit134" id="induction_of_hre_4x">induction of HRE(4x)</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- used cells: HEK stably transfected with HRE(4x)-CFP-CMV-mCherry, untransfected as a negative control<br/>
 
−
 
−
- induction in 24-well-plate<br/>
 
−
 
−
- add CoCl2: 0 µM, 20 µM, 40 µM, 80 µM, 100 µM, 320 µM, 640 µM<br/>
 
−
 
−
- FACS analysis<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_134'> </div>
 
−
<h2 class="sectionedit135" id="section021017">02.10.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_135'> </div>
 
−
<h3 class="sectionedit136" id="ph_measurements_with_hek_cells">pH measurements with HEK cells</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- 6-well-plate, 900 000 cells per well (90% confluency)<br/>
 
−
 
−
- DMEM (5 ml per well), pH set to 6.8<br/>
 
−
 
−
- after 6 h: pH=7.2<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_136'> </div>
 
−
<h3 class="sectionedit137" id="induction_of_hre">induction of HRE</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- used cells: Jurkat and HEK, stably transfected with HRE(4x)-CFP-CMV-mCherry<br/>
 
−
 
−
- induction with CoCl2: 0 µM, 50 µM, 100 µM, 150 µM, 200 µM, 250 µM, 300 µM, 350 µM<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_137'> </div>
 
−
<h3 class="sectionedit138" id="ph_measurements_with_hek_cells1">pH measurements with HEK cells</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- 6-well-plate, 900 000 cells per well<br/>
 
−
 
−
- DMEM (5 ml per well), pH set to 6.73<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_138'> </div>
 
−
<h2 class="sectionedit139" id="section031017">03.10.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_139'> </div>
 
−
<h3 class="sectionedit140" id="indcution_of_cre_4x_in_hek_cells">indcution of CRE(4x) in HEK cells</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- HEK stably transfected with CRE-CFP-CMV-mCherry, induction with acid (pH 6.7, 7.4, 8.1)<br/>
 
−
 
−
- FACS measurement failed<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_140'> </div>
 
−
<h3 class="sectionedit141" id="facs_analysis_of_cocl2_treated_hek_cells">FACS analysis of CoCl2 treated HEK cells</h3>
 
−
<div class="level3">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_141'> </div>
 
−
<h2 class="sectionedit142" id="section141017">14.10.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_142'> </div>
 
−
<h3 class="sectionedit143" id="repetition_of_the_interlab_study2">Repetition of the Interlab Study</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- transformation of positive control, negative control, Device 1-6
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_143'> </div>
 
−
<h2 class="sectionedit144" id="section151017">15.10.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_144'> </div>
 
−
<h3 class="sectionedit145" id="pick_colonies_of_the_interlab_study">pick colonies of the Interlab Study</h3>
 
−
<div class="level3">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_145'> </div>
 
−
<h2 class="sectionedit146" id="section171017">17.10.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_146'> </div>
 
−
<h3 class="sectionedit147" id="cell_measurements_of_the_interlab_study">cell measurements of the Interlab Study</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- OD600 and fluorescence intensity of each device after 0h, 2h, 4h and 6h<br/>
 
−
 
−
- again random fluorescence values<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_147'> </div>
 
−
<h3 class="sectionedit148" id="pei_transfection_of_cmv-vegfr2_sv40-tdag8">PEI transfection of CMV-VEGFR2, SV40-TDAG8</h3>
 
−
<div class="level3">
 
−
<div class="table sectionedit149"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">plasmid</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">CMV-VEGFR2</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">SV40-TDAG8</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_149'> </div><ol>
 
−
<li class="level1"><div class="li"> Split the cells 1:2 into 10 cm plate<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Thaw PEI reagent at RT<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> incubate 15 min at RT<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Add the mix to the culture<br/>
 
−
</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> incubate for 24 h<br/>
 
−
</div>
 
−
</li>
 
−
</ol>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_148'> </div>
 
−
<h2 class="sectionedit150" id="section181017">18.10.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_150'> </div>
 
−
<h3 class="sectionedit151" id="input_tests_in_hek_and_jurkat_cells">input tests in HEK and Jurkat cells</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>CRE(4x)-CFP-CMV-mCherry (stable), SV40-TDAG8 (transient):
 
−
</strong><br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
conditions:
 
−
</p>
 
−
<div class="table sectionedit152"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">pH</th><th class="col1">Forskolin + IBMX</th><th class="col2">other</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">6.5</td><td class="col1">-</td><td class="col2">-</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">7.1</td><td class="col1">-</td><td class="col2">-</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">7.7</td><td class="col1">-</td><td class="col2">-</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">no acid</td><td class="col1">Forskolin (100 µM) + IBMX (100 µM)</td><td class="col2">-</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">no acid</td><td class="col1">-</td><td class="col2">untransfected</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">no acid</td><td class="col1">-</td><td class="col2">without TDAG8</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_152'> </div>
 
−
<p>
 
−
<strong>Ctla4(4x)-CFP-CMV-mCherry (stable), CMV-VEGFR2 (transient):
 
−
</strong><br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
conditions:
 
−
</p>
 
−
<div class="table sectionedit153"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">VEGF (ng/ml)</th><th class="col1">Ionomycin (µmol/l)</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">0</td><td class="col1">-</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">25</td><td class="col1">-</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">50</td><td class="col1">-</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">100</td><td class="col1">-</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">-</td><td class="col1">1.25</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">-</td><td class="col1">2.5</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0">-</td><td class="col1">5.0</td>
 
−
</tr>
 
−
<tr class="row8">
 
−
<td class="col0">-</td><td class="col1">10.0</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_153'> </div>
 
−
<p>
 
−
<strong>HRE(4x)-CFP-CMV-mCherry (stable):
 
−
</strong><br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
conditions:
 
−
</p>
 
−
<div class="table sectionedit154"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">CoCl2 (µmol/ml)</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">50</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">100</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">200</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">400</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">600</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_154'> </div>
 
−
<p>
 
−
- fluorescence measurement after 0h, 1h, 2h, 4h, 6h, 9h, 16, 24h
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_151'> </div>
 
−
<h2 class="sectionedit155" id="section191017">19.10.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_155'> </div>
 
−
<h3 class="sectionedit156" id="hre_ctla4_and_hre_induction">HRE, Ctla4 and HRE induction</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
- medium change for Ctla4 and HRE cells:<br/>
 
−
 
−
- medium change for CRE:<br/>
 
−
 
−
- fluorescence measurement after 0h, 3h, 6h<br/>
 
−
 
−
</p>
 
−
<div class="tags"><span>
 
−
<a href="/igem2017/doku.php?id=tag:labbooks&amp;do=showtag&amp;tag=labbooks" class="wikilink1" title="tag:labbooks" rel="tag">labbooks</a>
 
−
</span></div>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_156'> </div>
 
−
    <!-- end rendered wiki content -->
 
−
    <div class="clearer"></div>
 
−
  </div>
 
−
  <!-- end div id bodyContent -->
 
−
 
−
 
 
−
 
−
 
−
</div>
 
−
<!-- end div id=content -->
 
−
 
−
 
−
 
−
</div>
 
−
</div>
 
−
</div>
 
−
</div>
 
−
</div>
 
−
 
−
<div class="container">
 
−
    <div class="row">
 
−
      <div class="col-md-12 text-center">
 
−
        <div class="flex-container">
 
−
          <div class="item">
 
−
<h1 class="sectionedit1" id="lab_notebook_cloning">Lab Notebook Cloning</h1>
 
−
<div class="level1">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_1'> </div>
 
−
<h2 class="sectionedit2" id="section19417">19.4.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_2'> </div>
 
−
<h3 class="sectionedit3" id="miniprep_of_pig17_003_4">Miniprep of pIG17_003/4</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Miniprep was performed with QIAprep Spin Miniprep Kit.
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_3'> </div>
 
−
<h3 class="sectionedit4" id="test_digestion_of_pig17_003_4">Test Digestion of pIG17_003/4</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>Reaction conditions</strong><br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
<strong>pIG17_003</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit5"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">14</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">EcoRI-HF</td><td class="col1">20U/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">CS-Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">DNA</td><td class="col1">264 ng/µl</td><td class="col2">3</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_5'> </div>
 
−
<p>
 
−
<strong>pIG17_004</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit6"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">15</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">EcoRI-HF</td><td class="col1">20U/µl</td><td class="col2">0.5</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">XbaI</td><td class="col1">20U/µl</td><td class="col2">0.5</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">CS-Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">334 ng/µl</td><td class="col2">2</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_6'> </div>
 
−
<p>
 
−
Electrophoresis failed and was repeated.
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_4'> </div>
 
−
<h2 class="sectionedit7" id="section20417">20.4.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_7'> </div>
 
−
<h3 class="sectionedit8" id="miniprep_of_pig17_001_2">Miniprep of pIG17_001/2</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Miniprep was performed with QIAprep Spin Miniprep Kit.
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_8'> </div>
 
−
<h2 class="sectionedit9" id="section21417">21.4.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_9'> </div>
 
−
<h3 class="sectionedit10" id="test_digestion_of_pig17_001_2_3_4">Test Digestion of pIG17_001/2/3/4</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>Reaction conditions</strong><br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
<strong>pIG17_001</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit11"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">14</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">EcoRI-HF</td><td class="col1">20U/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">CS-Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">DNA</td><td class="col1">178 ng/µl</td><td class="col2">3</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_11'> </div>
 
−
<p>
 
−
<strong>pIG17_002</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit12"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">15</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">EcoRI-HF</td><td class="col1">20U/µl</td><td class="col2">0.5</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">XbaI</td><td class="col1">20U/µl</td><td class="col2">0.5</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">CS-Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">246 ng/µl</td><td class="col2">2</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_12'> </div>
 
−
<p>
 
−
<strong>pIG17_003</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit13"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">14</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">EcoRI-HF</td><td class="col1">20U/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">CS-Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">DNA</td><td class="col1">156ng/µl</td><td class="col2">3</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_13'> </div>
 
−
<p>
 
−
<strong>pIG17_004</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit14"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">14</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">EcoRI-HF</td><td class="col1">20U/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">CS-Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">DNA</td><td class="col1">155ng/µl</td><td class="col2">3</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_14'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_10'> </div>
 
−
<h2 class="sectionedit15" id="section22417">22.4.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_15'> </div>
 
−
<h3 class="sectionedit16" id="miniprep_of_pig17_001_21">Miniprep of pIG17_001/2</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Miniprep was performed with QIAprep Spin Miniprep Kit.<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_16'> </div>
 
−
<h3 class="sectionedit17" id="sequencing_of_pig17_001_2">Sequencing of pIG17_001/2</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
sequencing returned successful
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_17'> </div>
 
−
<h2 class="sectionedit18" id="section23417">23.4.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_18'> </div>
 
−
<h3 class="sectionedit19" id="miniprep_of_pig17_001_22">Miniprep of pIG17_001/2</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Miniprep was performed with QIAprep Spin Miniprep Kit.<br/>
 
−
 
−
Plasmids were used for PEI transfection.
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_19'> </div>
 
−
<h2 class="sectionedit20" id="section25417">25.4.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_20'> </div>
 
−
<h3 class="sectionedit21" id="miniprep_of_pig17_008_9">Miniprep of pIG17_008/9</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Miniprep was performed with QIAprep Spin Miniprep Kit.<br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
DNA concentrations:<br/>
 
−
 
−
pIG17_008: 1043 ng/µl and 167,4 ng/µl <br/>
 
−
 
−
pIG17_009: 1295,1 ng/µl and 504,3 ng/µl <br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_21'> </div>
 
−
<h2 class="sectionedit22" id="section6517">6.5.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_22'> </div>
 
−
<h3 class="sectionedit23" id="test_digestion_of_pig17_008_9">Test Digestion of pIG17_008/9</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>Reaction conditions</strong><br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
<strong>pIG17_008</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit24"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">17</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">ClaI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">XbaI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">CS-Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">896ng/µl</td><td class="col2">0,5</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_24'> </div>
 
−
<p>
 
−
<strong>pIG17_009</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit25"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">17</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">PvuII</td><td class="col1">20U/µl</td><td class="col2">0,5</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">CS-Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">DNA</td><td class="col1">765ng/µl</td><td class="col2">0,5</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_25'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_23'> </div>
 
−
<h3 class="sectionedit26" id="pcr_of_pig17_009">PCR of pIG17_009</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>pIG17_009</strong>
 
−
Total reaction volume 25µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit27"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">15,75</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">oIG17_009 (Primer fw)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">oIG17_010 (Primer rv)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">5x Q5 Reaction Buffer</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">dNTPs</td><td class="col1">10mM</td><td class="col2">0,5</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">Polymerase Q5 HF</td><td class="col1">-</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0">DNA</td><td class="col1">10ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_27'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_26'> </div>
 
−
<h2 class="sectionedit28" id="section7517">7.5.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_28'> </div>
 
−
<h3 class="sectionedit29" id="test_digestion_of_pig17_008">Test Digestion of pIG17_008</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>Reaction conditions</strong><br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
<strong>pIG17_008</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit30"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">17</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">CalI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">KpnI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">CS-Buffer</td><td class="col1">10X</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">798ng/µl</td><td class="col2">0,5</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_30'> </div>
 
−
<p>
 
−
<strong>pIG17_008</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit31"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">17</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">PstI</td><td class="col1">20U/µl</td><td class="col2">0,5</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">CS-Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">DNA</td><td class="col1">886ng/µl</td><td class="col2">0,5</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_31'> </div>
 
−
<p>
 
−
<strong>pIG17_008</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit32"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">17</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">XbaI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">NdeI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">CS-Buffer</td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">µl</td><td class="col2">0,5</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_32'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_29'> </div>
 
−
<h3 class="sectionedit33" id="gel_extraction_of_pig17_009">Gel Extraction of pIG17_009</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Gel extraction was performed with QIAquick Gel Extraction Kit. <br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
Concentration of pIG17_009: 51,5 ng/µl <br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_33'> </div>
 
−
<h2 class="sectionedit34" id="section9517">9.5.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_34'> </div>
 
−
<h3 class="sectionedit35" id="digestion_of_pig17_002">Digestion of pIG17_002</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>Reaction conditions</strong><br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
<strong>pIG17_002</strong>
 
−
Total reaction volume 50µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit36"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">15</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">NdeI</td><td class="col1">20U/µl</td><td class="col2">1,5</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">AatII</td><td class="col1">20U/µl</td><td class="col2">1,5</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">CS-Buffer</td><td class="col1">10x</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">2µg</td><td class="col2">27</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_36'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_35'> </div>
 
−
<h2 class="sectionedit37" id="section13517">13.5.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_37'> </div>
 
−
<h3 class="sectionedit38" id="gel_extraction_of_pig17_002_--_seap_and_pa">Gel Extraction of pIG17_002 --&gt; SEAP and pA</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Gel extraction was performed with QIAquick Gel Extraction Kit. <br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
Concentration of pIG17_002: 84,5 ng/µl <br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_38'> </div>
 
−
<h3 class="sectionedit39" id="digestion_of_pig17_001_002">Digestion of pIG17_001/002</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>Reaction conditions</strong><br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
<strong>pIG17_001</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit40"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">13</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">NdeI</td><td class="col1">20U/µl</td><td class="col2">0,5</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">AatII</td><td class="col1">20U/µl</td><td class="col2">0,5</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">10x Fast Digest Green Buffer</td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">500ng</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_40'> </div>
 
−
<p>
 
−
undigested:
 
−
</p>
 
−
<div class="table sectionedit41"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">14</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">10x Fast Digest Green Buffer</td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">DNA</td><td class="col1">500ng</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_41'> </div>
 
−
<p>
 
−
<strong>pIG17_002</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit42"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">13</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">NdeI</td><td class="col1">20U/µl</td><td class="col2">0,5</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">AatII</td><td class="col1">20U/µl</td><td class="col2">0,5</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">10x Fast Digest Green Buffer</td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">500ng</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_42'> </div>
 
−
<p>
 
−
undigested:
 
−
</p>
 
−
<div class="table sectionedit43"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">14</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">10x Fast Digest Green Buffer</td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">DNA</td><td class="col1">500ng</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_43'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_39'> </div>
 
−
<h3 class="sectionedit44" id="pcr_of_pa_seap">PCR of pA/SEAP</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>pIG17_002</strong>
 
−
Total reaction volume 25µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit45"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">15,75</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">oIG17_012 (Primer fw)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">oIG17_013 (Primer rv)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">5x Q5 Reaction Buffer</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">dNTPs</td><td class="col1">10mM</td><td class="col2">0,5</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">Polymerase Q5 HF</td><td class="col1">-</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0">DNA</td><td class="col1">10ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_45'> </div>
 
−
<p>
 
−
<strong>pIG17_002</strong>
 
−
Total reaction volume 25µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit46"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">15,75</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">oIG17_014 (Primer fw)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">oIG17_015 (Primer rv)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">5x Q5 Reaction Buffer</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">dNTPs</td><td class="col1">10mM</td><td class="col2">0,5</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">Polymerase Q5 HF</td><td class="col1">-</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0">DNA</td><td class="col1">10ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_46'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_44'> </div>
 
−
<h3 class="sectionedit47" id="gel_ex_of_pa_seap">Gel Ex of pA/SEAP</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Gel extraction was performed with QIAquick Gel Extraction Kit. <br/>
 
−
 
−
Concentration of SEAP: 45,3 ng/µl <br/>
 
−
 
−
Concentration of pA: 49,1 ng/µl <br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_47'> </div>
 
−
<h2 class="sectionedit48" id="section15517">15.5.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_48'> </div>
 
−
<h3 class="sectionedit49" id="digestion_of_pig17_0021">Digestion of pIG17_002</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>Reaction conditions</strong><br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
<strong>pIG17_002</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit50"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">10</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">NdeI</td><td class="col1">20U/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">AatII</td><td class="col1">20U/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">CS-Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">105,3 ng/µl</td><td class="col2">6</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_50'> </div>
 
−
<p>
 
−
undigested:
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit51"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">15,5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">CS-Buffer</td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">DNA</td><td class="col1">-</td><td class="col2">2,5</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_51'> </div>
 
−
<p>
 
−
Because of the troubles to digest, the digestion was done for 2.5h by 37°C.
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_49'> </div>
 
−
<h3 class="sectionedit52" id="pcr_of_pig17_0091">PCR of pIG17_009</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>pIG17_009</strong>
 
−
Total rection volume 25µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit53"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">15,75</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">oIG17_009 (Primer fw)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">oIG17_010 (Primer rv)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">5x Q5 Reaction Buffer</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">dNTPs</td><td class="col1">10mM</td><td class="col2">0,5</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">Polymerase Q5 HF</td><td class="col1">-</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0">DNA</td><td class="col1">10ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_53'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_52'> </div>
 
−
<h2 class="sectionedit54" id="section18517">18.5.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_54'> </div>
 
−
<h3 class="sectionedit55" id="gibson_assembly_of_cmv_mcherry_pa_and_psb1c3">Gibson Assembly of CMV, mCherry, pA and pSB1C3</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>pIG17_058</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit56"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pSB1C3 Backbone</td><td class="col1">0,05pmol</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">CMV</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">mCherry</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pA</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_56'> </div>
 
−
<p>
 
−
Placed at 50°C for 1h
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_55'> </div>
 
−
<h3 class="sectionedit57" id="gibson_assembly_of_pig17_012_pig17_013_pig17_015_pig17_016">Gibson Assembly of pIG17_012, pIG17_013, pIG17_015, pIG17_016</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>pIG17_012</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit58"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pcDNA3.1</td><td class="col1">0,05pmol</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">HRE</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">SEAP</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pA</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_58'> </div>
 
−
<p>
 
−
Placed at 50°C for 1h
 
−
</p>
 
−
 
−
<p>
 
−
<strong>pIG17_013</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit59"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pcDNA3.1</td><td class="col1">0,05pmol</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">HRE</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">CFP</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pA</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_59'> </div>
 
−
<p>
 
−
Placed at 50°C for 1h
 
−
</p>
 
−
 
−
<p>
 
−
<strong>pIG17_015</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit60"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pcDNA3.1</td><td class="col1">0,05pmol</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">CRE</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">SEAP</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pA</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_60'> </div>
 
−
<p>
 
−
Placed at 50°C for 1h
 
−
</p>
 
−
 
−
<p>
 
−
<strong>pIG17_016</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit61"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pcDNA3.1</td><td class="col1">0,05pmol</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">CRE</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">eCFP</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pA</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_61'> </div>
 
−
<p>
 
−
Placed at 50°C for 1h
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_57'> </div>
 
−
<h3 class="sectionedit62" id="pcr_of_pig17_027_028_029">PCR of pIG17_027/028/029</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
PCR of extracted genomic DNA of Jurkat cells to amplify CTLA4
 
−
</p>
 
−
 
−
<p>
 
−
<strong>pIG17_027/028/029</strong>
 
−
Total rection volume 25µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit63"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">8</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">oIG17_048/049/050 (Primer fw) (1000bp/380bp/330bp)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">oIG17_051 (Primer rv)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">5x Q5 MM</td><td class="col1">-</td><td class="col2">12,5</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">10ng/µl</td><td class="col2">2</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_63'> </div>
 
−
<p>
 
−
Annealing temperatures:<br/>
 
−
 
−
CTLA4 1000bp: 66°C<br/>
 
−
 
−
CTLA4 380bp: 64°C<br/>
 
−
 
−
CTLA4 330bp: 65°C<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_62'> </div>
 
−
<h3 class="sectionedit64" id="gel_picture">Gel picture</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:ctla4_gel.jpg" class="media" title="notebooks:ctla4_gel.jpg"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=02ac5a&amp;media=notebooks:ctla4_gel.jpg" class="media" alt="" width="400" /></a> <br/>
 
−
 
−
<br/>
 
−
 
−
CTLA4 380bp and 330bp have the expected length. For CTLA4 1000bp we couldn&#039;t see any band on the gel.
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_64'> </div>
 
−
<h3 class="sectionedit65" id="gel_extraction_of_pig17_028_029">Gel Extraction of pIG17_028/029</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
CTLA4 1000bp was not extracted because it couldn&#039;t be seen on the gel.<br/>
 
−
 
−
Gel extraction was performed with QIAquick Gel Extraction Kit. <br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_65'> </div>
 
−
<h2 class="sectionedit66" id="section19517">19.5.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_66'> </div>
 
−
<h3 class="sectionedit67" id="gibson_assembly_of_cmv_mcherry_delta_psti_pa_and_psb1c3">Gibson Assembly of CMV, mCherry delta PstI, pA and pSB1C3</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>pIG17_058</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit68"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pSB1C3 Backbone</td><td class="col1">0,05pmol</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">CMV</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">mCherry delta PstI</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pA</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_68'> </div>
 
−
<p>
 
−
Placed at 50°C for 1h
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_67'> </div>
 
−
<h2 class="sectionedit69" id="section21517">21.5.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_69'> </div>
 
−
<h3 class="sectionedit70" id="subcloning_of_ctla4_330_380">Subcloning of CTLA4_330/380</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>330</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit71"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">T4 Ligase </td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">T4 Ligase Reaction buffer</td><td class="col1">2x</td><td class="col2">10</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">pJET2.1</td><td class="col1">50ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">5ng/µl</td><td class="col2">5</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_71'> </div>
 
−
<p>
 
−
<strong>380</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit72"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">T4 Ligase </td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">T4 Ligase Reaction buffer</td><td class="col1">2x</td><td class="col2">10</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">pJET2.1</td><td class="col1">50ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">5ng/µl</td><td class="col2">5</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_72'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_70'> </div>
 
−
<h3 class="sectionedit73" id="sequencing_of_pig17_012_13_15_16">Sequencing of pIG17_012/13/15/16</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Sequencing returned positive
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_73'> </div>
 
−
<h2 class="sectionedit74" id="section22517">22.5.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_74'> </div>
 
−
<h3 class="sectionedit75" id="test_digestion_of_mcherry_and_the_gibson_assembly_g11">Test digestion of mCherry and the Gibson Assembly G1.1</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Digestion of the constructs was performed to test the function of the enzymes.<br/>
 
−
 
−
<br/>
 
−
 
−
<strong>Reaction conditions</strong><br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
<strong>mCherry</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit76"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">13,75</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">KpnI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">CS-Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">DNA</td><td class="col1">-</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_76'> </div>
 
−
<p>
 
−
undigested:
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit77"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">14</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">CS-Buffer</td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">DNA</td><td class="col1">-</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_77'> </div>
 
−
<p>
 
−
undigested without CS-Buffer:
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit78"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">16</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">DNA</td><td class="col1">-</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_78'> </div>
 
−
<p>
 
−
<strong>Gibson Assembly G1.1</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit79"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">12,25</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">HindIII</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">AflII</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">ClaI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">CS-Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">DNA</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_79'> </div>
 
−
<p>
 
−
undigested:
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit80"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">13</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">CS-Buffer</td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">DNA</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_80'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_75'> </div>
 
−
<h3 class="sectionedit81" id="gel_picture1">Gel picture</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:test_digestion_gibson_5_22_17.jpg" class="media" title="notebooks:test_digestion_gibson_5_22_17.jpg"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=9d969b&amp;media=notebooks:test_digestion_gibson_5_22_17.jpg" class="media" alt="" width="400" /></a>
 
−
</p>
 
−
 
−
<p>
 
−
Because of the lack of an AafII restriction site in the G1.1 construct there are only two bands to be seen. <br/>
 
−
 
−
All bands have the expected length and the enzymes still work.
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_81'> </div>
 
−
<h2 class="sectionedit82" id="section23517">23.5.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_82'> </div>
 
−
<h3 class="sectionedit83" id="test_digestion_of_ctla4_380bp_and_330bp">Test digestion of CTLA4 380bp and 330bp</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>Reaction conditions</strong><br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
<strong>CTLA4 330 (3,4) and CTLA4 380 (1)</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit84"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">12,5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">NotI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">NcoI-HF</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">CS-Buffer</td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">500ng</td><td class="col2">5</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_84'> </div>
 
−
<p>
 
−
<strong>CTLA4 330 (1,2) and CTLA4 380 (3,4)</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit85"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">13,5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">NotI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">NcoI-HF</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">CS-Buffer</td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">500ng</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_85'> </div>
 
−
<p>
 
−
<strong>CTLA4 380 (2)</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit86"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">14,5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">NotI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">NcoI-HF</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">CS-Buffer</td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">500ng</td><td class="col2">3</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_86'> </div>
 
−
<p>
 
−
Undigested samples were done, respectively.<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_83'> </div>
 
−
<h3 class="sectionedit87" id="gel_picture2">Gel picture</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:test_digestion_ctla4_5_23_17.jpg" class="media" title="notebooks:test_digestion_ctla4_5_23_17.jpg"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=9f4638&amp;media=notebooks:test_digestion_ctla4_5_23_17.jpg" class="media" alt="" width="400" /></a><br/>
 
−
 
−
CTLA4 330bp number 1 and also number 1 of CTLA4 380bp were chosen for further procedures. <br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_87'> </div>
 
−
<h2 class="sectionedit88" id="section30517">30.5.17</h2>
 
−
<div class="level2">
 
−
 
−
<p>
 
−
Obtaining pSAM200 and pMF111 form AG Weber
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_88'> </div>
 
−
<h2 class="sectionedit89" id="section31517">31.5.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_89'> </div>
 
−
<h2 class="sectionedit90" id="test_digestion_of_psam200_pmf111">Test digestion of pSAM200/pMF111</h2>
 
−
<div class="level2">
 
−
 
−
<p>
 
−
<strong>pSAM200</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit91"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">13,5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">XbaI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">XhoI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">CS-Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">170,3ng/µl</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_91'> </div>
 
−
<p>
 
−
<strong>pMF200</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit92"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">13,5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">XbaI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">XhoI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">CS-Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">133,7ng/µl</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_92'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_90'> </div>
 
−
<h2 class="sectionedit93" id="section3617">3.6.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_93'> </div>
 
−
<h3 class="sectionedit94" id="midiprep_of_pig17_003_082">Midiprep of pIG17_003/082</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Midiprep was performed with the Promega PureYield Plasmid Midiprep System Kit.
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_94'> </div>
 
−
<h3 class="sectionedit95" id="miniprep_of_ctla4_330bp_380bp">Miniprep of CTLA4 (330bp, 380bp)</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Miniprep was performed with QIAprep Spin Miniprep Kit.
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_95'> </div>
 
−
<h3 class="sectionedit96" id="extension_pcr_of_ctla4_330bp_380bp">Extension PCR of CTLA4 (330bp, 380bp)</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>CTLA4 (330bp)</strong><br/>
 
−
 
−
Total rection volume 25µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit97"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">9</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">oIG17_105 (Primer fw)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">oIG17_106 (Primer rv)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">5x Q5 MM</td><td class="col1">-</td><td class="col2">12,5</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">10ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_97'> </div>
 
−
<p>
 
−
<strong>CTLA4 (380bp)</strong><br/>
 
−
 
−
Total rection volume 25µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit98"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">9</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">oIG17_107 (Primer fw)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">oIG17_106 (Primer rv)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">5x Q5 MM</td><td class="col1">-</td><td class="col2">12,5</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">10ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_98'> </div>
 
−
<p>
 
−
Annealing temperatures:<br/>
 
−
 
−
CTLA4 380bp: 59°C<br/>
 
−
 
−
CTLA4 330bp: 65°C<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_96'> </div>
 
−
<h2 class="sectionedit99" id="section4617">4.6.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_99'> </div>
 
−
<h3 class="sectionedit100" id="extension_pcr_of_ctla4_330bp_380bp1">Extension PCR of CTLA4 (330bp, 380bp)</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Because the extension PCR didn&#039;t work the day before, we performed another one.<br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
<strong>CTLA4 (330bp)</strong><br/>
 
−
 
−
Total rection volume 25µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit101"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">9</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">oIG17_105 (Primer fw)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">oIG17_106 (Primer rv)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">5x Q5 MM</td><td class="col1">-</td><td class="col2">12,5</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">10ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_101'> </div>
 
−
<p>
 
−
<strong>CTLA4 (380bp)</strong><br/>
 
−
 
−
Total rection volume 25µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit102"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">9</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">oIG17_107 (Primer fw)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">oIG17_106 (Primer rv)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">5x Q5 MM</td><td class="col1">-</td><td class="col2">12,5</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">10ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_102'> </div>
 
−
<p>
 
−
Annealing temperatures:<br/>
 
−
 
−
CTLA4 380bp: 59°C<br/>
 
−
 
−
CTLA4 330bp: 65°C<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_100'> </div>
 
−
<h3 class="sectionedit103" id="gel_extraction_of_ctla4_330bp_380bp_of_the_extension_pcr">Gel Extraction of CTLA4 (330bp, 380bp) of the extension PCR</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Gel extraction was performed with QIAquick Gel Extraction Kit. <br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_103'> </div>
 
−
<h3 class="sectionedit104" id="gibson_assembly_of_ctla4_gfp_seap_pa_and_bb_-_pig17_019_020_022_023">Gibson Assembly of CTLA4, GFP/SEAP, pA and BB - pIG17_019/020/022/023</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>CTLA4 (330bp) and GFP - pIG17_023</strong><br/>
 
−
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit105"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pSB1C3 Backbone</td><td class="col1">0,06pmol</td><td class="col2">0,75</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">CTLA4 (330bp)</td><td class="col1">0,06pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">GFP</td><td class="col1">0,06pmol</td><td class="col2">0,5</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pA</td><td class="col1">0,06pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">H2O</td><td class="col1">-</td><td class="col2">1,75</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_105'> </div>
 
−
<p>
 
−
<strong>CTLA4 (330bp) and SEAP - pIG17_020</strong><br/>
 
−
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit106"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pSB1C3 Backbone</td><td class="col1">0,06pmol</td><td class="col2">0,75</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">CTLA4 (330bp)</td><td class="col1">0,06pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">SEAP</td><td class="col1">0,06pmol</td><td class="col2">0,75</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pA</td><td class="col1">0,06pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">H2O</td><td class="col1">-</td><td class="col2">1,5</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_106'> </div>
 
−
<p>
 
−
<strong>CTLA4 (380bp) and GFP - pIG17_022</strong><br/>
 
−
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit107"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pSB1C3 Backbone</td><td class="col1">0,06pmol</td><td class="col2">0,75</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">CTLA4 (380bp)</td><td class="col1">0,06pmol</td><td class="col2">0,5</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">GFP</td><td class="col1">0,06pmol</td><td class="col2">0,5</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pA</td><td class="col1">0,06pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">H2O</td><td class="col1">-</td><td class="col2">2,25</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_107'> </div>
 
−
<p>
 
−
<strong>CTLA4 (380bp) and SEAP - pIG17_019</strong><br/>
 
−
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit108"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pSB1C3 Backbone</td><td class="col1">0,06pmol</td><td class="col2">0,75</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">CTLA4 (380bp)</td><td class="col1">0,06pmol</td><td class="col2">0,5</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">SEAP</td><td class="col1">0,06pmol</td><td class="col2">0,75</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pA</td><td class="col1">0,06pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">H2O</td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_108'> </div>
 
−
<p>
 
−
Placed at 50°C for 1h
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_104'> </div>
 
−
<h3 class="sectionedit109" id="transformation_pig17_019_020_022_023_030">Transformation pIG17_019/020/022/023/030</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Transformation was done with DH5alpha Z-competent cells for all CTLA4-GFP/SEAP Gibson assemblies.<br/>
 
−
 
−
pIG17_030 was done again because there were no colonies on the dish.
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_109'> </div>
 
−
<h2 class="sectionedit110" id="section5617">5.6.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_110'> </div>
 
−
<h3 class="sectionedit111" id="midiprep_of_pig17_004_008_009">Midiprep of pIG17_004/008/009</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Midiprep was performed with the Promega PureYield Plasmid Midiprep System Kit.
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_111'> </div>
 
−
<h3 class="sectionedit112" id="gel">Gel</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Because of the trouble to get a transformation of pIG17_030, the Gibson assembly and the used constructs were loaded on a gel, in order to find out where the problem lies.
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_112'> </div>
 
−
<h3 class="sectionedit113" id="result_of_gibson_assembly">Result of Gibson assembly</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
CTLA4 330 with GFP did growth<br/>
 
−
 
−
CTLA4 380 with GFP did growth<br/>
 
−
 
−
CTLA4 330 with SEAP did not growth<br/>
 
−
 
−
CTLA4 380 with SEAP did not growth<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_113'> </div>
 
−
<h2 class="sectionedit114" id="section6617">6.6.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_114'> </div>
 
−
<h3 class="sectionedit115" id="extention_pcr_of_luc">Extention PCR of LUC</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Extention PCR made to get rid of unwanted the <em class="u">cutting site</em> <br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_115'> </div>
 
−
<h3 class="sectionedit116" id="miniprep_of_ctla4_gfp_pig17022_pig17023">Miniprep of CTLA4_GFP (pIG17.022, pIG17.023)</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Miniprep with JenaBioscience. The yield was too poor to work with. New cultures are needed. o/n are done.
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_116'> </div>
 
−
<h3 class="sectionedit117" id="sequencing_of_seap">Sequencing of SEAP</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<em class="u">SEAP construct (19, 20)</em> are send to sequencing.
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_117'> </div>
 
−
<h3 class="sectionedit118" id="sequencing_analysis_of_pcre_and_phre_pig17006_pig17007">Sequencing analysis of pCRE and pHRE (pIG17.006, pIG17.007)</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<em class="u">The results:</em>
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_118'> </div>
 
−
<h3 class="sectionedit119" id="pick_of_ctla4_330_seap_pig17020">Pick of CTLA4 330_SEAP (pIG17.020)</h3>
 
−
<div class="level3">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_119'> </div>
 
−
<h3 class="sectionedit120" id="test_digest_of_ctla4_330_and_380_seap_gfp_pig17019_pig17020_pig17022_pig17023">Test digest of CTLA4 330 and 380 SEAP &amp; GFP (pIG17.019, pIG17.020, pIG17.022, pIG17.023)</h3>
 
−
<div class="level3">
 
−
<div class="table sectionedit121"><table class="inline">
 
−
<tr class="row0">
 
−
<td class="col0 leftalign"> Construct  </td><td class="col1"> </td><td class="col2">Enzym </td><td class="col3">Size </td><td class="col4"> Size undigested</td>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">CTLA4 330 </td><td class="col1">GFP </td><td class="col2">HindI II </td><td class="col3 leftalign">2210bp, 925bp  </td><td class="col4">3127pb </td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0"> </td><td class="col1">SEAP </td><td class="col2">HindI II </td><td class="col3 rightalign">        2210pb, 1726pb</td><td class="col4">3928pb </td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">CTLA4 380 </td><td class="col1">GFP </td><td class="col2"> HindI II</td><td class="col3">2260pb, 929pb </td><td class="col4">3177pb </td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0"> </td><td class="col1">SEAP </td><td class="col2"> HindI II</td><td class="col3 rightalign">        2260pb, 1726pb</td><td class="col4">3978pb </td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_121'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_120'> </div>
 
−
<h3 class="sectionedit122" id="transformation_of_hre_seap_pig17030">Transformation of HRE_SEAP (pIG17.030)</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Transformation made with 5μl. Did not worked.
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_122'> </div>
 
−
<h3 class="sectionedit123" id="test_digest_of_luceferase">Test digest of Luceferase</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Concentration: 462ng/μl
 
−
</p>
 
−
<div class="table sectionedit124"><table class="inline">
 
−
<tr class="row0">
 
−
<td class="col0"> </td><td class="col1"> Volumina for digest </td><td class="col2"> Volumina for negative control </td>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">DNA</td><td class="col1">1,1μl </td><td class="col2">1,1μl </td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">Buffer</td><td class="col1">2μl</td><td class="col2"> 2μl</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">Water</td><td class="col1">16,4μl</td><td class="col2">16,9μl</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">Enzyme 1</td><td class="col1">0,25μl</td><td class="col2"> </td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">Enzyme 2</td><td class="col1">0,25μl</td><td class="col2"> </td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">Total</td><td class="col1 leftalign">20μl  </td><td class="col2">20μl</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_124'> </div>
 
−
<p>
 
−
Used enzyme: 1:XbaI: 37°C, 2:NotI:37°C
 
−
Buffer: NEBuffer 3.1
 
−
DNA used: 500ng
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_123'> </div>
 
−
<h3 class="sectionedit125" id="test_digest_of_pig17019_pig17020_pig17022_pig17023">Test digest of pIG17.019, pIG17.020, pIG17.022, pIG17.023</h3>
 
−
<div class="level3">
 
−
<div class="table sectionedit126"><table class="inline">
 
−
<tr class="row0">
 
−
<td class="col0"> </td><td class="col1">330 GFP</td><td class="col2">330 SEAP </td><td class="col3">380 GFP </td><td class="col4">330 SEAP </td><td class="col5">380 SEAP </td>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">DNA </td><td class="col1">250ng </td><td class="col2">250ng </td><td class="col3">250ng </td><td class="col4">250ng </td><td class="col5">250ng </td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">Water </td><td class="col1"> </td><td class="col2"> </td><td class="col3"> </td><td class="col4"> </td><td class="col5"></td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">Enzyme </td><td class="col1">0,5μl </td><td class="col2">0,5μl </td><td class="col3">0,5μl </td><td class="col4">0,5μl </td><td class="col5">0,5μl </td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">Buffer </td><td class="col1">2μl </td><td class="col2">2μl </td><td class="col3">2μl </td><td class="col4">2μl </td><td class="col5">2μl </td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">Total</td><td class="col1">20μl</td><td class="col2">20μl</td><td class="col3">20μl</td><td class="col4">20μl</td><td class="col5">20μl</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_126'> </div>
 
−
<p>
 
−
<em class="u">On the gel:                    </em>
 
−
Loading dye: 5μl
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_125'> </div>
 
−
<h3 class="sectionedit127" id="midi_prep_of_cmv_pig17008_pig17009">Midi Prep of CMV (pIG17.008, pIG17.009)</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Midiprep done with Promega PureYield™ Plasmid Midiprep System kit
 
−
</p>
 
−
<div class="table sectionedit128"><table class="inline">
 
−
<tr class="row0">
 
−
<td class="col0"> </td><td class="col1"> 1.elution</td><td class="col2">2.elution</td>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">pIG17.008</td><td class="col1">142,1ng/μl</td><td class="col2">50,1ng/μl</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pIG17.009</td><td class="col1">89,3ng/μl</td><td class="col2">64,1ng/μl</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_128'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_127'> </div>
 
−
<h2 class="sectionedit129" id="section7617">7.6.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_129'> </div>
 
−
<h3 class="sectionedit130" id="miniprep_of_pig17_019_020_022_023">Miniprep of pIG17_019/020/022/023</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Miniprep was performed with Jena Bioscience Kit.
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_130'> </div>
 
−
<h3 class="sectionedit131" id="results">Results</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Transformation of HRE SEAP with mCherry didn&#039;t grow
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_131'> </div>
 
−
<h3 class="sectionedit132" id="test_digestof_pig17_019_020_022_023">Test digestof pIG17_019/020/022/023</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Test diget with HindIII<br/>
 
−
 
−
DNA concentration of 500ng/μl (4μl of DNA)
 
−
10xPuffer (2μl)
 
−
H2O till 20μl in the PCR tube.
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_132'> </div>
 
−
<h3 class="sectionedit133" id="gatc_sequencing">GATC sequencing</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
3x HIF1-a with 30ng/μl with the primer oIG17_119 (3μl with 26μl H2O)
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_133'> </div>
 
−
<h2 class="sectionedit134" id="section8617">8.6.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_134'> </div>
 
−
<h3 class="sectionedit135" id="trouble_shooting_of_the_promega_midiprep_kit">Trouble shooting of the Promega Midiprep kit</h3>
 
−
<div class="level3">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_135'> </div>
 
−
<h3 class="sectionedit136" id="extention_pcr_with_mcherry_and_pig17_012">Extention PCR with mCherry and pIG17_012</h3>
 
−
<div class="level3">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_136'> </div>
 
−
<h2 class="sectionedit137" id="section9617">9.6.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_137'> </div>
 
−
<h3 class="sectionedit138" id="test_digestion_of_pig17_019_020_022_023">Test Digestion of pIG17_019/020/022/023</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>Reaction conditions</strong><br/>
 
−
 
−
</p>
 
−
 
−
<p>
 
−
pIG17_019/020:
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit139"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">17,5 - x</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">Nde I</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">Cla I</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">CS-Buffer</td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">500 ng</td><td class="col2">x</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_139'> </div>
 
−
<p>
 
−
<br/>
 
−
 
−
pIG17_022/023:
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit140"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">17,5 - x</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">Nco I</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">Cla I</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">CS-Buffer</td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">500 ng</td><td class="col2">x</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_140'> </div>
 
−
<p>
 
−
Undigested: <br/>
 
−
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit141"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">18 - x</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">CS-Buffer</td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">DNA</td><td class="col1">500 ng</td><td class="col2">x</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_141'> </div>
 
−
<p>
 
−
<br/>
 
−
 
−
Incubation for 1h at 37 degrees
 
−
<br/>
 
−
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_138'> </div>
 
−
<h3 class="sectionedit142" id="gel_extraction_of_pig17_012_and_mcherry">Gel extraction of pIG17_012 and mCherry</h3>
 
−
<div class="level3">
 
−
<div class="table sectionedit143"><table class="inline">
 
−
<tr class="row0">
 
−
<td class="col0">DNA</td><td class="col1">Gel</td><td class="col2">QG</td><td class="col3">Isopropanol</td>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">pIG17_012</td><td class="col1">0,12g</td><td class="col2">240μl</td><td class="col3">81,16</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">mCherry</td><td class="col1">0,08g</td><td class="col2">360μl</td><td class="col3">122,4μl</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_143'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_142'> </div>
 
−
<h3 class="sectionedit144" id="midiprep_of_pig17_008_and_pig17_009">Midiprep of pIG17_008 and pIG17_009</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
2 bands on the gel : Contamination!
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_144'> </div>
 
−
<h2 class="sectionedit145" id="section14617">14.6.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_145'> </div>
 
−
<h3 class="sectionedit146" id="test_digestion_of_sdm_of_luc">Test Digestion of SDM of LUC</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>SDM of LUC</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit147"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">13,75</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">BspEI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">Tango Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">DNA</td><td class="col1">151,7ng/µl</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_147'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_146'> </div>
 
−
<h3 class="sectionedit148" id="gibson_assembly_pig17_31_34_37_86">Gibson assembly pIG17_31/34/37/86</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>pIG17_31</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit149"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pcDNA3.1</td><td class="col1">0,05pmol</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">pIG17_012</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">pIG17_058</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_149'> </div>
 
−
<p>
 
−
<strong>pIG17_34</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit150"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pcDNA3.1</td><td class="col1">0,05pmol</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">pIG17_013</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">pIG17_058</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_150'> </div>
 
−
<p>
 
−
<strong>pIG17_37</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit151"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pcDNA3.1</td><td class="col1">0,05pmol</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">pIG17_015</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">pIG17_058</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_151'> </div>
 
−
<p>
 
−
<strong>pIG17_86</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit152"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pcDNA3.1</td><td class="col1">0,05pmol</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">pIG17_016</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">pIG17_058</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_152'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_148'> </div>
 
−
<h2 class="sectionedit153" id="section12617">12.6.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_153'> </div>
 
−
<h3 class="sectionedit154" id="midiprep_of_pig17_008_009">Midiprep of pIG17_008/009</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Midiprep was performed with the Promega Kit.
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_154'> </div>
 
−
<h3 class="sectionedit155" id="gibson_assembly_of_luciferase_with_sdm">Gibson Assembly of Luciferase with SDM</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Total rection volume 24µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit156"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">12</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">Luciferase Backbone</td><td class="col1">0,06pmol</td><td class="col2">7,5</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">PCR product 1 of Luciferase</td><td class="col1">0,06pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">PCR product 2 of Luciferase</td><td class="col1">0,06pmol</td><td class="col2">2,5</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">PCR product 3 of Luciferase</td><td class="col1">0,05pmol</td><td class="col2">0,5</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">Water</td><td class="col1">-</td><td class="col2">0,5</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_156'> </div>
 
−
<p>
 
−
Placed at 50°C for 1h
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_155'> </div>
 
−
<h2 class="sectionedit157" id="section6717">6.7.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_157'> </div>
 
−
<h3 class="sectionedit158" id="mini_prep_of_hif1_-_alpha_fusion_proteins_pmf111_psb1c3_luc_delta_bspei">Mini Prep of HIF1 - alpha fusion proteins (pmf111/psb1c3/LUC delta BspEI)</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Miniprep done with Promega PureYield™ Plasmid Miniprep System kit
 
−
</p>
 
−
<div class="table sectionedit159"><table class="inline">
 
−
<tr class="row0">
 
−
<td class="col0"> </td><td class="col1"> concentration</td>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">pmf111</td><td class="col1">48,1ng/μl</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">psb1c3</td><td class="col1">107,6ng/μl</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">LUC delta BspEI </td><td class="col1"> 289,6ng/μl </td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_159'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_158'> </div>
 
−
<h3 class="sectionedit160" id="miniprep_of_pig17_003_41">Miniprep of pIG17_003/4</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Miniprep was not performed due to the fact that the overnight culture got spilled out.
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_160'> </div>
 
−
<h3 class="sectionedit161" id="pcr_of_hif1_-_alpha_fusion_proteins_psb1c3_luc_delta_bspei">PCR of HIF1 - alpha fusion proteins (psb1c3/LUC delta BspEI)</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>psb1c3</strong><br/>
 
−
 
−
Total rection volume 25µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit162"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">9</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">oIG17_198 (Primer fw)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">oIG17_197 (Primer rv)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">5x Q5 MM</td><td class="col1">-</td><td class="col2">12,5</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">10ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_162'> </div>
 
−
<p>
 
−
<strong>LUC delta BspEI</strong><br/>
 
−
 
−
Total rection volume 25µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit163"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">9</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">oIG17_101 (Primer fw)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">oIG17_202 (Primer rv)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">5x Q5 MM</td><td class="col1">-</td><td class="col2">12,5</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">10ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_163'> </div>
 
−
<p>
 
−
<strong>LUC delta BspEI</strong><br/>
 
−
 
−
Total rection volume 25µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit164"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">9</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">oIG17_203 (Primer fw)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">oIG17_100 (Primer rv)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">5x Q5 MM</td><td class="col1">-</td><td class="col2">12,5</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">10ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_164'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_161'> </div>
 
−
<h2 class="sectionedit165" id="section17617">17.6.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_165'> </div>
 
−
<h3 class="sectionedit166" id="testdigestion_of_pig17_31_34_37_86">Testdigestion of pIG17_31/34/37/86</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>pIG17_031</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit167"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">13,75</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">BspEI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">KpnI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">Tango Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">120,7ng/µl</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_167'> </div>
 
−
<p>
 
−
<strong>pIG17_034</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit168"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">13,75</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">BspEI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">KpnI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">Tango Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">130,5ng/µl</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_168'> </div>
 
−
<p>
 
−
<strong>pIG17_037</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit169"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">13,75</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">BspEI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">KpnI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">Tango Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">111,7ng/µl</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_169'> </div>
 
−
<p>
 
−
<strong>pIG17_087</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit170"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">13,75</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">BspEI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">KpnI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">Tango Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">101,9ng/µl</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_170'> </div>
 
−
<p>
 
−
Positive clones were send to sequencing
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_166'> </div>
 
−
<h2 class="sectionedit171" id="section18617">18.6.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_171'> </div>
 
−
<h3 class="sectionedit172" id="gibson_assembly_of_pig17_063_064_065">Gibson Assembly of pIG17_063/064/065</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>pIG17_063</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit173"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pcDNA3.1</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">HRE</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">LUC</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pA</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">pIG17_058</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_173'> </div>
 
−
<p>
 
−
<strong>pIG17_064</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit174"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pcDNA3.1</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">CRE</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">LUC</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pA</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">pIG17_058</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_174'> </div>
 
−
<p>
 
−
<strong>pIG17_065</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit175"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pcDNA3.1</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">NFAT b.s.</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">LUC</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pA</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">pIG17_058</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_175'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_172'> </div>
 
−
<h2 class="sectionedit176" id="section220617">22.06.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_176'> </div>
 
−
<h3 class="sectionedit177" id="test_digestion_of_pig17_063_064_065">Test digestion of pIG17_063/064/065</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>pIG17_063</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit178"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">13,75</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">BspEI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">KpnI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">Tango Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">100,7ng/µl</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_178'> </div>
 
−
<p>
 
−
<strong>pIG17_064</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit179"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">13,75</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">BspEI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">KpnI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">Tango Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">120,5ng/µl</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_179'> </div>
 
−
<p>
 
−
<strong>pIG17_064</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit180"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">13,75</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">BspEI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">KpnI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">Tango Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">98,1ng/µl</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_180'> </div>
 
−
<p>
 
−
Positive clones were sent to sequencing
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_177'> </div>
 
−
<h2 class="sectionedit181" id="section28617">28.6.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_181'> </div>
 
−
<h3 class="sectionedit182" id="digestion_of_pig17_31_34_37_86_for_ligation_into_transferplasmid">Digestion of pIG17_31/34/37/86 for ligation into Transferplasmid</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>pIG17_031</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit183"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">13,75</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">ClaI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">KpnI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">Tango Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">120,1ng/µl</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_183'> </div>
 
−
<p>
 
−
<strong>pIG17_034</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit184"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">13,75</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">ClaI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">KpnI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">Tango Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">160,8ng/µl</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_184'> </div>
 
−
<p>
 
−
<strong>pIG17_037</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit185"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">13,75</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">ClaI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">KpnI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">Tango Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">143,3ng/µl</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_185'> </div>
 
−
<p>
 
−
<strong>pIG17_086</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit186"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">13,75</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">ClaI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">KpnI</td><td class="col1">20U/µl</td><td class="col2">0,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">Tango Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">198,5ng/µl</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_186'> </div>
 
−
<p>
 
−
<strong>Transferplasmid</strong>
 
−
Total reaction volume 50µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit187"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">30,25</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">BspEI</td><td class="col1">20U/µl</td><td class="col2">0,5</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">KpnI</td><td class="col1">20U/µl</td><td class="col2">0,5</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">Tango Buffer</td><td class="col1">10x</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">300,4ng/µl</td><td class="col2">4</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_187'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_182'> </div>
 
−
<h3 class="sectionedit188" id="t4_ligation_of_pig17_31_34_37_87_into_transfer_plasmid">T4 Ligation of pIG17_31/34/37/87 into Transfer plasmid</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong> pIG17_31 </strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit189"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">T4 Ligase </td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">T4 Ligase Reaction buffer</td><td class="col1">2x</td><td class="col2">10</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">Transfer plasmid</td><td class="col1">70ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pIG17_031</td><td class="col1">13ng/µl</td><td class="col2">5</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_189'> </div>
 
−
<p>
 
−
<strong> pIG17_34 </strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit190"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">T4 Ligase </td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">T4 Ligase Reaction buffer</td><td class="col1">2x</td><td class="col2">10</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">Transfer plasmid</td><td class="col1">70ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pIG17_034</td><td class="col1">15ng/µl</td><td class="col2">5</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_190'> </div>
 
−
<p>
 
−
<strong> pIG17_37 </strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit191"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">T4 Ligase </td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">T4 Ligase Reaction buffer</td><td class="col1">2x</td><td class="col2">10</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">Transfer plasmid</td><td class="col1">70ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pIG17_037</td><td class="col1">13ng/µl</td><td class="col2">5</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_191'> </div>
 
−
<p>
 
−
<strong> pIG17_87 </strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit192"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">T4 Ligase </td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">T4 Ligase Reaction buffer</td><td class="col1">2x</td><td class="col2">10</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">Transfer plasmid</td><td class="col1">70 ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pIG17_087</td><td class="col1">5ng/µl</td><td class="col2">5</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_192'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_188'> </div>
 
−
<h2 class="sectionedit193" id="section2717">2.7.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_193'> </div>
 
−
<h3 class="sectionedit194" id="gibson_assembly_of_hre-gfp-pa-sv40-neomycin_zeozin">Gibson Assembly of HRE-GFP-pA-SV40-Neomycin/Zeozin</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>HRE-GFP-pA-SV40-Neomycin</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit195"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pcDNA3.1</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">pIg17_013</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">SV40</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">Neomycin</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">pA</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_195'> </div>
 
−
<p>
 
−
<strong>HRE-GFP-pA-SV40-Zeozinin</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit196"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pcDNA3.1</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">pIg17_013</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">SV40</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">Zeocin</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">pA</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_196'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_194'> </div>
 
−
<h2 class="sectionedit197" id="section030717">03.07.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_197'> </div>
 
−
<h3 class="sectionedit198" id="gibson_assembly_of_sv40-tetr-pa">Gibson assembly of SV40-TetR-pA</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>HRE-GFP-pA-SV40-Neomycin</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit199"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pcDNA3.1</td><td class="col1">0,05pmol</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">SV40</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">VP16-TetR</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pA</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_199'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_198'> </div>
 
−
<h2 class="sectionedit200" id="section7717">7.7.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_200'> </div>
 
−
<h3 class="sectionedit201" id="mini_prep_of_pig17_031_034_086_119">Mini Prep of  pIG17_031/034/086/119</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Miniprep done with Promega PureYield™ Plasmid Miniprep System kit
 
−
</p>
 
−
<div class="table sectionedit202"><table class="inline">
 
−
<tr class="row0">
 
−
<td class="col0"> </td><td class="col1"> concentration</td>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">pIG17.031</td><td class="col1">46,6ng/μl</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pIG17.034</td><td class="col1">45,7ng/μl</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">pIG17.086</td><td class="col1">47,9ng/μl</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">pIG17.119</td><td class="col1">390,8ng/μl</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_202'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_201'> </div>
 
−
<h3 class="sectionedit203" id="pcr_of_hif1_-_alpha_fusion_proteins_pmf111_psb1c3_standard_part_we_got_from_igem">PCR of HIF1 - alpha fusion proteins (pMF111/pSB1C3(standard part we got from iGEM))</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>pMF111 #1</strong><br/>
 
−
 
−
Total rection volume 25µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit204"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">9</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">oIG17_177 (Primer fw)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">oIG17_211 (Primer rv)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">5x Q5 MM</td><td class="col1">-</td><td class="col2">12,5</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">10ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_204'> </div>
 
−
<p>
 
−
<strong>pMF111 #3</strong><br/>
 
−
 
−
Total rection volume 25µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit205"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">9</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">oIG17_150 (Primer fw)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">oIG17_176 (Primer rv)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">5x Q5 MM</td><td class="col1">-</td><td class="col2">12,5</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">10ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_205'> </div>
 
−
<p>
 
−
<strong>pSB1C3 #2</strong><br/>
 
−
 
−
Total rection volume 25µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit206"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">9</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">oIG17_198 (Primer fw)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">oIG17_197 (Primer rv)</td><td class="col1">10µM</td><td class="col2">1,25</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">5x Q5 MM</td><td class="col1">-</td><td class="col2">12,5</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">DNA</td><td class="col1">10ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_206'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_203'> </div>
 
−
<h3 class="sectionedit207" id="gel_picture3">Gel picture</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:gel_pic.png" class="media" title="notebooks:gel_pic.png"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=d02abb&amp;media=notebooks:gel_pic.png" class="media" alt="" width="400" /></a>
 
−
</p>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_207'> </div>
 
−
<h3 class="sectionedit208" id="midiprep_of_31_34_86_119">Midiprep of 31, 34, 86, 119</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Midiprep with Promega Midiprep kit<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit209"><table class="inline">
 
−
<tr class="row0">
 
−
<td class="col0">pIG17_031</td><td class="col1">6,0ng/μl </td><td class="col2"></td>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">pIG17_034</td><td class="col1">4,4ng/μl </td><td class="col2"></td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pIG17_086 </td><td class="col1 leftalign">9,4ng/μl  </td><td class="col2">6,9ng/μl </td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">pIG17_119 </td><td class="col1 leftalign">156ng/μl  </td><td class="col2">167ng/μl </td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_209'> </div><div class="tags"><span>
 
−
<a href="/igem2017/doku.php?id=tag:labbooks&amp;do=showtag&amp;tag=labbooks" class="wikilink1" title="tag:labbooks" rel="tag">labbooks</a>
 
−
</span></div>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_208'> </div>
 
−
<h2 class="sectionedit210" id="section8717">8.7.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_210'> </div>
 
−
<h3 class="sectionedit211" id="gibson_assembly_of_teto_hre_cre_nfat_bs-hif_hif-luc-cmv-mcherry">Gibson Assembly of TetO/HRE/CRE/NFAT b.s.-HIF/HIF-LUC-CMV-mCherry</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>TetO-HIF-LUC-pA-CMV-mCherry</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit212"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">6</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pcDNA3.1</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">TetO</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">HIF</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">LUC</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">pA</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0">pIG17_058</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_212'> </div>
 
−
<p>
 
−
<strong>HRE-HIF-pA-CMV-mCherry</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit213"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pcDNA3.1</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">HRE</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">HIF</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pA</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">pIG17_058</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_213'> </div>
 
−
<p>
 
−
<strong>CRE-HIF-pA-CMV-mCherry</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit214"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pcDNA3.1</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">CRE</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">HIF</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pA</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">pIG17_058</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_214'> </div>
 
−
<p>
 
−
<strong>NFAT bs 330-HIF-pA-CMV-mCherry</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit215"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pcDNA3.1</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">NFAT b.s. 330</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">HIF</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pA</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">pIG17_058</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_215'> </div>
 
−
<p>
 
−
<strong>NFAT bs 380-HIF-pA-CMV-mCherry</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit216"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pcDNA3.1</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">NFAT b.s. 380</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">HIF</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pA</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">pIG17_058</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_216'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_211'> </div>
 
−
<h2 class="sectionedit217" id="section9717">9.7.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_217'> </div>
 
−
<h3 class="sectionedit218" id="bacterial_glycerol_stocks_for_long-term_storage_of_plasmids">Bacterial Glycerol Stocks for Long-term Storage of Plasmids</h3>
 
−
<div class="level3">
 
−
<ul>
 
−
<li class="level1"><div class="li"> pIG17_037</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> pIG17_086</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> pIG17_119</div>
 
−
</li>
 
−
</ul>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_218'> </div>
 
−
<h3 class="sectionedit219" id="mini_prep_of_pig17_118_120_121">Mini Prep of  pIG17_118/120/121</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Miniprep done with Promega PureYield™ Plasmid Miniprep System kit
 
−
</p>
 
−
<div class="table sectionedit220"><table class="inline">
 
−
<tr class="row0">
 
−
<td class="col0"> </td><td class="col1"> concentration</td>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">pIG17_118</td><td class="col1">51,6ng/μl</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pIG17_120 #1</td><td class="col1">50ng/μl</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">pIG17_120 #2</td><td class="col1">56ng/μl</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">pIG17_121 #1</td><td class="col1">208,3ng/μl</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pIG17_121 #2</td><td class="col1">56ng/μl</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_220'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_219'> </div>
 
−
<h2 class="sectionedit221" id="section10717">10.7.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_221'> </div>
 
−
<h3 class="sectionedit222" id="mini_prep_of_pig17_31_zeomycin_and_neomycin_pig17_097">Mini Prep of  pIG17_31 zeomycin and neomycin/pIG17_097</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Miniprep done with Promega PureYield™ Plasmid Miniprep System kit
 
−
</p>
 
−
<div class="table sectionedit223"><table class="inline">
 
−
<tr class="row0">
 
−
<td class="col0"> </td><td class="col1"> concentration</td>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">pIG17_031 zeo</td><td class="col1">107,9ng/μl</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pIG17_031 neo</td><td class="col1">25,7ng/μl</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">pIG17_097 1 #1</td><td class="col1">30,8ng/μl</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">pIG17_097 1 #2</td><td class="col1">34,2ng/μl</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pIG17_097 2 #1</td><td class="col1">38,4ng/μl</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">pIG17_097 2 #2</td><td class="col1">40,0ng/μl</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0">pIG17_097 2 #3</td><td class="col1">32,3ng/μl</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_223'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_222'> </div>
 
−
<h2 class="sectionedit224" id="section12717">12.7.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_224'> </div>
 
−
<h3 class="sectionedit225" id="mini_prep_of_teto-luc-hif_teto-hif-luc_pig17_031_tetr">Mini Prep of  TetO-Luc-HIF/TetO-HIF-Luc/pIG17_031 TetR</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Miniprep done with Promega PureYield™ Plasmid Miniprep System kit
 
−
</p>
 
−
<div class="table sectionedit226"><table class="inline">
 
−
<tr class="row0">
 
−
<td class="col0"> </td><td class="col1"> concentration</td>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">pIG17_031 TetR</td><td class="col1">22,0ng/μl</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">TetO-Luc-HIF #1</td><td class="col1">32,4ng/μl</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">TetO-HIF-Luc #1</td><td class="col1">174,5ng/μl</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_226'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_225'> </div>
 
−
<h2 class="sectionedit227" id="section13717">13.7.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_227'> </div>
 
−
<h3 class="sectionedit228" id="mini_prep_of_pcre-hif_ctla4-hif">Mini Prep of  pCRE-HIF/CTLA4-HIF</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Miniprep done with Promega PureYield™ Plasmid Miniprep System kit
 
−
</p>
 
−
<div class="table sectionedit229"><table class="inline">
 
−
<tr class="row0">
 
−
<td class="col0"> </td><td class="col1"> concentration</td>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">pCRE-HIF #3</td><td class="col1">28,3ng/μl</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pCRE-HIF #2</td><td class="col1">30,1ng/μl</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">pCRE-HIF #1</td><td class="col1">30,4ng/μl</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">CTLA4-HIF(380) #1</td><td class="col1">32,1ng/μl</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">CTLA4-HIF(380) #2</td><td class="col1">33,1ng/μl</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">CTLA4-HIF(380) #3</td><td class="col1">32,8ng/μl</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0">CTLA4-HIF(330) #2</td><td class="col1">29,9ng/μl</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_229'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_228'> </div>
 
−
<h2 class="sectionedit230" id="section15717">15.7.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_230'> </div>
 
−
<h3 class="sectionedit231" id="ligation_of_hre_4_cre_4_nfat_bs_4_into_pig17_031">Ligation of HRE*4/CRE*4/NFAT bs*4 into pIG17_031</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>HRE*4</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit232"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">6</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">T4 Ligase </td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">T4 Ligase Reaction buffer</td><td class="col1">2x</td><td class="col2">10</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">pIG17_031</td><td class="col1">65ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">HRE*4</td><td class="col1">10ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_232'> </div>
 
−
<p>
 
−
<strong>CRE*4</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit233"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">6</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">T4 Ligase </td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">T4 Ligase Reaction buffer</td><td class="col1">2x</td><td class="col2">10</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">pIG17_031</td><td class="col1">65ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">CRE*4</td><td class="col1">10ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_233'> </div>
 
−
<p>
 
−
<strong>NFAT bs*4</strong>
 
−
Total reaction volume 20µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit234"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">H20</td><td class="col1">-</td><td class="col2">6</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">T4 Ligase </td><td class="col1">-</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">T4 Ligase Reaction buffer</td><td class="col1">2x</td><td class="col2">10</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">pIG17_031</td><td class="col1">65ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">NFAT bs*4</td><td class="col1">10ng/µl</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_234'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_231'> </div>
 
−
<h2 class="sectionedit235" id="section16717">16.7.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_235'> </div>
 
−
<h3 class="sectionedit236" id="gibson_assembly_of_hre_4_cre_4_nfat_bs_4-cfp-cmv-mcherry">Gibson Assembly of HRE*4/CRE*4/NFAT bs*4-CFP-CMV-mCherry</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
<strong>HRE*4-CFP-CMV-mCherry</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit237"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">Transfer vector</td><td class="col1">0,05pmol</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">HRE*4</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">CFP</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pIG17_058</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_237'> </div>
 
−
<p>
 
−
<strong>CRE*4-CFP-CMV-mCherry</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit238"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">Transfer vector</td><td class="col1">0,05pmol</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">CRE*4</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">CFP</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pIG17_058</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_238'> </div>
 
−
<p>
 
−
<strong>NFAT bs*4-CFP-CMV-mCherry</strong>
 
−
Total rection volume 10µl<br/>
 
−
 
−
</p>
 
−
<div class="table sectionedit239"><table class="inline">
 
−
<thead>
 
−
<tr class="row0">
 
−
<th class="col0">component</th><th class="col1">concentration</th><th class="col2">volume [µL]</th>
 
−
</tr>
 
−
</thead>
 
−
<tr class="row1">
 
−
<td class="col0">2x Gibson MM</td><td class="col1">-</td><td class="col2">5</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">Transfer vector</td><td class="col1">0,05pmol</td><td class="col2">2</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">NFAT bs*4</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">CFP</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pIG17_058</td><td class="col1">0,05pmol</td><td class="col2">1</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_239'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_236'> </div>
 
−
<h2 class="sectionedit240" id="section19717">19.7.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_240'> </div>
 
−
<h3 class="sectionedit241" id="mini_prep_of_pcre-hif_ctla4-hif_pig17_096">Mini Prep of  pCRE-HIF/CTLA4-HIF/pIG17_096</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Miniprep done with Promega PureYield™ Plasmid Miniprep System kit
 
−
</p>
 
−
<div class="table sectionedit242"><table class="inline">
 
−
<tr class="row0">
 
−
<td class="col0"> </td><td class="col1"> concentration</td>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">pCRE-HIF</td><td class="col1">26,7ng/μl</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pCRE-HIF</td><td class="col1">187,7ng/μl</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">pCRE-HIF</td><td class="col1">153,0ng/μl</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">pCRE-HIF</td><td class="col1">102,0ng/μl</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">CTLA4-HIF(380)</td><td class="col1">32,4ng/μl</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">CTLA4-HIF(380)</td><td class="col1">340,1ng/μl</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0">CTLA4-HIF(380)</td><td class="col1">273,5ng/μl</td>
 
−
</tr>
 
−
<tr class="row8">
 
−
<td class="col0">CTLA4-HIF(380)</td><td class="col1">70,4ng/μl</td>
 
−
</tr>
 
−
<tr class="row9">
 
−
<td class="col0">CTLA4-HIF(380)</td><td class="col1">88,81ng/μl</td>
 
−
</tr>
 
−
<tr class="row10">
 
−
<td class="col0">CTLA4-HIF(380)</td><td class="col1">59,9ng/μl</td>
 
−
</tr>
 
−
<tr class="row11">
 
−
<td class="col0">CTLA4-HIF(330)</td><td class="col1">29,0ng/μl</td>
 
−
</tr>
 
−
<tr class="row12">
 
−
<td class="col0">CTLA4-HIF(330)</td><td class="col1">315,2ng/μl</td>
 
−
</tr>
 
−
<tr class="row13">
 
−
<td class="col0">CTLA4-HIF(330)</td><td class="col1">98,8ng/μl</td>
 
−
</tr>
 
−
<tr class="row14">
 
−
<td class="col0">CTLA4-HIF(330)</td><td class="col1">344,6ng/μl</td>
 
−
</tr>
 
−
<tr class="row15">
 
−
<td class="col0">CTLA4-HIF(330)</td><td class="col1">135,1ng/μl</td>
 
−
</tr>
 
−
<tr class="row16">
 
−
<td class="col0">pIG17_096</td><td class="col1">342,8ng/μl</td>
 
−
</tr>
 
−
<tr class="row17">
 
−
<td class="col0">pIG17_096</td><td class="col1">31,3ng/μl</td>
 
−
</tr>
 
−
<tr class="row18">
 
−
<td class="col0">pIG17_096</td><td class="col1">71,3ng/μl</td>
 
−
</tr>
 
−
<tr class="row19">
 
−
<td class="col0">pIG17_096</td><td class="col1">99,7ng/μl</td>
 
−
</tr>
 
−
<tr class="row20">
 
−
<td class="col0">pIG17_096</td><td class="col1">55,8ng/μl</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_242'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_241'> </div>
 
−
<h3 class="sectionedit243" id="sequencing_of_pcre-hif_ctla4-hif_330_ctla4-hif_330_pig17_096_pig17_097_pig17_118_pig17_120">Sequencing of  pCRE-HIF/CTLA4-HIF 330/CTLA4-HIF 330/pIG17_096/pIG17_097/pIG17_118/pIG17_120</h3>
 
−
<div class="level3">
 
−
<div class="table sectionedit244"><table class="inline">
 
−
<tr class="row0">
 
−
<td class="col0">plasmid number</td><td class="col1"> primer</td>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">pCRE-HIF</td><td class="col1">oIG17_166/018/164/174/175/119</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">CTLA4-HIF(380)</td><td class="col1">oIG17_166/018/164/174/175/119</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">CTLA4-HIF(330)</td><td class="col1">oIG17_166/018/164/174/175/119</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">pIG17_096</td><td class="col1">oIG17_174/175/169/170/018</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pIG17_097</td><td class="col1">oIG17_054</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">pIG17_118</td><td class="col1">oIG17_019</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0">pIG17_120</td><td class="col1">oIG17_019</td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_244'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_243'> </div>
 
−
<h2 class="sectionedit245" id="section21717">21.7.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_245'> </div>
 
−
<h3 class="sectionedit246" id="mini_prep_of_hre_x_mult-gfp-cmv-mcherry_cre_x_mult-gfp-cmv-mcherry_hre_ecfp_zeo_tetr_vp16_neo">Mini Prep of  HRE x Mult-GFP-CMV-mCherry/CRE x Mult-GFP-CMV-mCherry/HRE ecFP zeo/TetR VP16 neo</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Miniprep done with Promega PureYield™ Plasmid Miniprep System kit
 
−
</p>
 
−
<div class="table sectionedit247"><table class="inline">
 
−
<tr class="row0">
 
−
<td class="col0"> </td><td class="col1">concentration</td><td class="col2">curve</td>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">HRE x Mult-GFP-CMV-mCherry #1</td><td class="col1">39,4ng/μl</td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:hre_ecfp3.jpg" class="media" title="notebooks:hre_ecfp3.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=87081b&amp;media=notebooks:hre_ecfp3.jpg" class="media" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">HRE x Mult-GFP-CMV-mCherry #2</td><td class="col1">28,9ng/μl</td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:hre_x_mult2.jpg" class="media" title="notebooks:hre_x_mult2.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=95a04d&amp;media=notebooks:hre_x_mult2.jpg" class="media" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">HRE x Mult-GFP-CMV-mCherry #3</td><td class="col1">28,9ng/μl</td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:hre-mult3.jpg" class="media" title="notebooks:hre-mult3.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=663f96&amp;media=notebooks:hre-mult3.jpg" class="media" alt="" width="200" /></a> </td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">HRE x Mult-GFP-CMV-mCherry #4</td><td class="col1">44,6ng/μl</td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:hre_x_mult4.jpg" class="media" title="notebooks:hre_x_mult4.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=d0d6f1&amp;media=notebooks:hre_x_mult4.jpg" class="media" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">CRE x Mult-GFP-CMV-mCherry #1</td><td class="col1">18,9ng/μl</td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:20170721_125058.jpg" class="media" title="notebooks:20170721_125058.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=934095&amp;media=notebooks:20170721_125058.jpg" class="media" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">CRE x Mult-GFP-CMV-mCherry #2</td><td class="col1">49,8ng/μl</td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:cre_x_mult2.jpg" class="media" title="notebooks:cre_x_mult2.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=d3706b&amp;media=notebooks:cre_x_mult2.jpg" class="media" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0">CRE x Mult-GFP-CMV-mCherry #3</td><td class="col1">20,9ng/μl</td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:cre_x_mult.jpg" class="media" title="notebooks:cre_x_mult.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=9b570d&amp;media=notebooks:cre_x_mult.jpg" class="media" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row8">
 
−
<td class="col0">CRE x Mult-GFP-CMV-mCherry #4</td><td class="col1">25,2ng/μl</td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:cre_x_mult4.jpg" class="media" title="notebooks:cre_x_mult4.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=b40b4b&amp;media=notebooks:cre_x_mult4.jpg" class="media" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row9">
 
−
<td class="col0">HRE ecFP zeo #1</td><td class="col1">35,2ng/μl</td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:hre_ecfp1.jpg" class="media" title="notebooks:hre_ecfp1.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=3c5f23&amp;media=notebooks:hre_ecfp1.jpg" class="media" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row10">
 
−
<td class="col0">HRE ecFP zeo #2</td><td class="col1">26,4ng/μl</td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:hre_ecfp2.jpg" class="media" title="notebooks:hre_ecfp2.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=003759&amp;media=notebooks:hre_ecfp2.jpg" class="media" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row11">
 
−
<td class="col0">HRE ecFP zeo #3</td><td class="col1">36,2ng/μl</td><td class="col2"></td>
 
−
</tr>
 
−
<tr class="row12">
 
−
<td class="col0">HRE ecFP zeo #4</td><td class="col1">17,3ng/μl</td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:hre_ecfp4.jpg" class="media" title="notebooks:hre_ecfp4.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=d7d245&amp;media=notebooks:hre_ecfp4.jpg" class="media" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row13">
 
−
<td class="col0">TetR VP16 neo #1</td><td class="col1">21,7ng/μl</td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:tetr_1.jpg" class="media" title="notebooks:tetr_1.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=ff66b1&amp;media=notebooks:tetr_1.jpg" class="media" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row14">
 
−
<td class="col0">TetR VP16 neo #2</td><td class="col1">30,4ng/μl</td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:tetr_2.jpg" class="media" title="notebooks:tetr_2.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=b3fa1a&amp;media=notebooks:tetr_2.jpg" class="media" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row15">
 
−
<td class="col0">TetR VP16 neo #3</td><td class="col1">66,7ng/μl</td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:tetr3.jpg" class="media" title="notebooks:tetr3.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=ee60b5&amp;media=notebooks:tetr3.jpg" class="media" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row16">
 
−
<td class="col0">TetR VP16 neo #4</td><td class="col1">28,1ng/μl</td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:tetr4.jpg" class="media" title="notebooks:tetr4.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=5987fb&amp;media=notebooks:tetr4.jpg" class="media" alt="" width="200" /></a></td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_247'> </div><ul>
 
−
<li class="level1"><div class="li"> we also went upstairs to check our concentrations with Toolbox Nanodrop but there wasn&#039;t much difference </div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Denis asked Nicole if she can do a Miniprep for us with her kit</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Nicole suggested to try to use the buffer instead of water to see if it will change something </div>
 
−
</li>
 
−
</ul>
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_246'> </div>
 
−
<h2 class="sectionedit248" id="section24717">24.7.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_248'> </div>
 
−
<h3 class="sectionedit249" id="mini_prep_of_pig17_012_013_015_016">Mini Prep of  pIG17_012/013/015/016</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Miniprep done with Promega PureYield™ Plasmid Miniprep System kit
 
−
</p>
 
−
<div class="table sectionedit250"><table class="inline">
 
−
<tr class="row0">
 
−
<td class="col0"> </td><td class="col1">concentration</td><td class="col2">curve</td>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">pIG17_012</td><td class="col1">187,1ng/μl</td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:012.jpg" class="media" title="notebooks:012.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=8b1de4&amp;media=notebooks:012.jpg" class="media" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pIG17_013</td><td class="col1">53,9ng/μl</td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:015.jpg" class="media" title="notebooks:015.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=849c2c&amp;media=notebooks:015.jpg" class="media" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">pIG17_015</td><td class="col1">49,1ng/μl</td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:019.jpg" class="media" title="notebooks:019.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=cca3c7&amp;media=notebooks:019.jpg" class="media" alt="" width="200" /></a></td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">pIG17_016</td><td class="col1">26,1ng/μl</td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:020.jpg" class="media" title="notebooks:020.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=077b58&amp;media=notebooks:020.jpg" class="media" alt="" width="200" /></a></td>
 
−
</tr>
 
−
</table></div>
 
−
<div class='secedit editbutton_table editbutton_250'> </div>
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_249'> </div>
 
−
<h2 class="sectionedit251" id="section29717">29.7.17</h2>
 
−
<div class="level2">
 
−
 
−
</div>
 
−
<div class='secedit editbutton_section editbutton_251'> </div>
 
−
<h3 class="sectionedit252" id="mini_prep_of_pig17_003_031_119">Mini Prep of  pIG17_003/031/119/</h3>
 
−
<div class="level3">
 
−
 
−
<p>
 
−
Miniprep done with Promega PureYield™ Plasmid Miniprep System kit (in 40μl Wasser eluiert)
 
−
</p>
 
−
<div class="table sectionedit253"><table class="inline">
 
−
<tr class="row0">
 
−
<td class="col0"> </td><td class="col1">concentration</td>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">pIG17_003</td><td class="col1">16,9ng/μl</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">pIG17_003</td><td class="col1">90,8ng/μl</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">pIG17_003</td><td class="col1">144,0ng/μl</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">pIG17_004</td><td class="col1">154,9ng/μl</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">pIG17_004</td><td class="col1">280,7ng/μl</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">pIG17_031</td><td class="col1">541,1ng/μl (!)</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0">pIG17_031</td><td class="col1">257,1ng/μl (!)</td>
 
−
</tr>
 
−
<tr class="row8">
 
−
<td class="col0">pIG17_031</td><td class="col1">336,1ng/μl (!)</td>
 
−
</tr>
 
−
<tr class="row9">
 
−
<td class="col0">pIG17_034</td><td class="col1">23,0ng/μl (!)</td>
 
−
</tr>
 
−
<tr class="row10">
 
−
<td class="col0">pIG17_119</td><td class="col1">318,6ng/μl (!)</td>
 
−
</tr>
 
−
<tr class="row11">
 
−
<td class="col0">pIG17_119</td><td class="col1">19,3ng/μl</td>
 
−
</tr>
 
−
<tr class="row12">
 
−
<td class="col0">pIG17_119</td><td class="col1">31,9ng/μl</td>
 
−
</tr>
 
−
<tr class="row13">
 
−
<td class="col0">pIG17_119</td><td class="col1">12,1ng/μl</td>
 
−
</tr>
 
−
<tr class="row14">
 
−
<td class="col0">pIG17_119</td><td class="col1">53,2ng/μl</td>
 
−
</tr>
 
−
<tr class="row15">
 
−
<td class="col0">pIG17_119</td><td class="col1">314,0ng/μl (!)</td>
 
−
</tr>
 
−
<tr class="row16">
 
−
<td class="col0">HRE GFP</td><td class="col1">109,9ng/μl</td>
 
−
</tr>
 
−
<tr class="row17">
 
−
<td class="col0">HRE GFP</td><td class="col1">43,9ng/μl</td>
 
−
</tr>
 
−
<tr class="row18">
 
−
<td class="col0">HRE GFP</td><td class="col1">89,9ng/μl</td>
 
−
</tr>
 
−
<tr class="row19">
 
−
<td class="col0">HRE GFP</td><td class="col1">147,1ng/μl</td>
 
−
</tr>
 
−
<tr class="row20">
 
−
<td class="col0">HRE GFP</td><td class="col1">104,3ng/μl</td>
 
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<td class="col0">HRE GFP</td><td class="col1">67,9ng/μl (!)</td>
 
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<td class="col0">HRE GFP</td><td class="col1">125,2ng/μl</td>
 
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<td class="col0">HRE GFP</td><td class="col1">153,1ng/μl</td>
 
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<td class="col0">HRE GFP</td><td class="col1">318,2ng/μl</td>
 
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<td class="col0">HRE GFP</td><td class="col1">294,9ng/μl</td>
 
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<td class="col0">HRE GFP</td><td class="col1">148,6ng/μl</td>
 
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<td class="col0">HRE GFP</td><td class="col1">82,1ng/μl</td>
 
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<td class="col0">HRE GFP</td><td class="col1">423,9ng/μl (!)</td>
 
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<td class="col0">HRE GFP</td><td class="col1">427,2ng/μl (!)</td>
 
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<h3 class="sectionedit255" id="mini_prep_of_pig17_503_tet-gfp">Mini Prep of  pIG17_503/Tet-GFP</h3>
 
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Miniprep done with Promega PureYield™ Plasmid Miniprep System kit (in 40μl Wasser eluiert)
 
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<td class="col0">pIG17_503</td><td class="col1">242,6ng/μl</td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:20542808_1382915745120230_1869775090_o.jpg" class="media" title="notebooks:20542808_1382915745120230_1869775090_o.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=90b992&amp;media=notebooks:20542808_1382915745120230_1869775090_o.jpg" class="media" alt="" width="200" /></a></td>
 
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<td class="col0">Tet-GFP</td><td class="col1">255,1ng/μl</td><td class="col2"><a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aph&amp;media=notebooks:20536001_1382915755120229_915165855_o.jpg" class="media" title="notebooks:20536001_1382915755120229_915165855_o.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=1e7a9f&amp;media=notebooks:20536001_1382915755120229_915165855_o.jpg" class="media" alt="" width="200" /></a></td>
 
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Latest revision as of 21:38, 31 October 2017

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