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>
+
−
 
+
−
             
+
−
              <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>
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<h3 class="sectionedit283" id="pei_transfection_of_hek_cells_for_lenvirus_production1">PEI transfection of HEK cells for lenvirus production</h3>
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Constructs used: pIG17_130, CRE 4x <br/>
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<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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<h2 class="sectionedit284" id="section200917">20.09.17</h2>
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<h3 class="sectionedit285" id="pei_transfection_of_hek_cells_for_lenvirus_production2">PEI transfection of HEK cells for lenvirus production</h3>
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Constructs used: NFAT 4x, HRE 4x <br/>
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<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>
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The concentrated viruses were stored in -80°C freezer as backup.<br/>
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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>
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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/>
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All samples were done in triplicates<br/>
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Induction with CoCl2, the used concentrations of CoCl2 were: 0, 20, 40, 80, 160, 320 and 640 µM.<br/>
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<h3 class="sectionedit291" id="seap_assay1">SEAP assay</h3>
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The SEAP assay was performed as before, this time we included a cell count of each pooled triplicate. <br/>
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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.
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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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We could see that only 20, 40 and 80 µM CoCl2 showed a steeper slope in our samples than the 0 µM control.
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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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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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<h3 class="sectionedit295" id="hre_testing_with_stable_hek_cell_lines">HRE testing with stable HEK cell lines</h3>
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<h2 class="sectionedit296" id="section041017">04.10.17</h2>
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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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<p>
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Constructs used: pIG17_133, Suiside plasmid
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<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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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/>
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<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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There is a clear shift and our cells are really CFP positive and not GFP positive
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<h3 class="sectionedit300" id="cre_test_in_jurkat_cells">CRE Test in Jurkat cells</h3>
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<p>
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<strong>With Forskolin and IBMX, the cAMP pathway in the cell is activated.</strong>
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<ul>
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<li class="level1"><div class="li"> Cell lines: WT Jurkat, stable CRE4X Jurkat</div>
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</li>
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<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>
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<p>
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FACS: <br/>
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Cells were gated for living, single, mCherry positive cells and then the amount of CFP positive cells was measured: <br/>
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<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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<li class="level1"><div class="li"> On 11th of Oct.: PEI transfection with CMV-TDAG8 plasmid. </div>
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We obtained the following results after gating for living, single, mCherry positive cells:
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Latest revision as of 21:38, 31 October 2017

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