Difference between revisions of "Team:BOKU-Vienna/Notebook"

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<!---TEST--->
 
<!---TEST--->
 +
<body>
 +
<div id="mydiv">
 +
  <h4>Monday, July 3rd.</h4>
 +
  <ul class="mylist">
 +
    <li>Team discussion and overview:</li>
 +
      <ul>
 +
        <li>Christina, Felicia, Josef, Lisa, Magdalena, Mathias, Melanie, Michael, Mina, Rebecca, Valentin, Julian</li>
 +
      </ul>
 +
    <li>Planning of the procedures including primer design</li>
 +
    <li><a href="https://2017.igem.org/Team:BOKU-Vienna/Protocol#lbmedium1" target="_blank">Preparing the growth media and autoclaving</a></li>
 +
      <ul><li>LB medium preparation (4L): Mina, Michael</li></ul>
 +
      <ul><li>Autoclaving: Mina, Rebecca</li></ul>
 +
  </ul>
 +
</div>
 +
 +
<div id="date-7-4"> 
 +
  <h1>Tuesday, July 4th.</h1>
 +
<ul>
 +
    <li>Planning and fundraising:</li>
 +
      <ul>
 +
        <li>Christina, Felicia, Josef, Lisa, Martin, Mathias, Melanie, Michael, Mina, Rebecca, Valentin, Julian</li>
 +
      </ul>
 +
    <li>Preparation of growth media:</li>
 +
      <ul><li>20 plates <a href="https://2017.igem.org/Team:BOKU-Vienna/Protocol#lbmedium1" target="_blank">LB medium</a> + chloramphenicol: Mina, Lisa, Mathias</li></ul>
 +
      <ul><li>2 flasks <a href="https://2017.igem.org/Team:BOKU-Vienna/Protocol#lbagar" target="_blank">LB agar</a>: Mina, Lisa, Mathias</li></ul>
 +
    <li>Making cells from straind DH10B chemically competent as a control for the InterLab Study</li> 
 +
</ul>
 +
</div>
 +
 +
<div id="date-7-5">
 +
  <h1>Wednesday, July 5th.</h1>
 +
  <ul>
 +
    <li>Planning and fundraising:</li>
 +
      <ul>
 +
        <li>Christina, Felicia, Josef, Lisa, Magdalena, Martin, Mathias, Melanie, Michael, Mina, Rebecca, Julian</li>
 +
      </ul>
 +
    <li>Aliquoting 1mL LB into Eppendorf tubes</li>
 +
        <ul><li>Lisa, Josef</li></ul>
 +
    <li>Transferring Backbones onto agar plates:</li>
 +
        <ul><li>Julian</li></ul>
 +
    <li>Interlab challenge - transformation: According to <a href="https://2017.igem.org/Team:BOKU-Vienna/Protocol#transformation" target="_blank">protocol</a>:</li>
 +
        <ul>Lisa, Mathias, Michael, Mina, Rebecca</ul>
 +
        <ul><li>8 transformations - from <a href="https://2017.igem.org/Team:BOKU-Vienna/Protocol#interlab" target="_blank">Kit Plate 6 InterLab</a></li></ul>
 +
        <ul><li>8 plasmids plated onto agar medium: Positive control (50µL, 200µL), Negative Control (50µL, 200µL), Transformant (20µL, 100µL, 200µL), Test device 1-6 (50µL, 200µL)</li></ul>
 +
        <ul><li>stored at 35°C</li></ul>
 +
  </ul>
 +
</div>
 +
 +
<div id="date-7-6">
 +
  <h1>Thursday, July 6th.</h1>
 +
  <ul>
 +
    <li>Team discussion, planning and fundraising</li>
 +
    <li>Counting colonies of Transformants for the Interlab Challenge:</li>
 +
      <ul><li>Colonies 200µL: 77, 20µL: 26, 100µL: 43</li></ul>
 +
      <img src="https://static.igem.org/mediawiki/2017/1/17/T--BOKU-Vienna--Notebook_countedplate1.jpg" alt="Transformants on plate" height="200">
 +
      <img src="https://static.igem.org/mediawiki/2017/f/f6/T--BOKU-Vienna--Notebook_countedplate2.jpg" alt="Transformants on plate" height="200">
 +
      <img src="https://static.igem.org/mediawiki/2017/2/2c/T--BOKU-Vienna--Notebook_countedplate3.jpg" alt="Transformants on plate" height="200">
 +
    <li>Miniprep of 8 plasmids for Golden Gate Standard - according to <a href="https://2017.igem.org/Team:BOKU-Vienna/Protocol#miniprep" target="_blank">protocol</a></li>
 +
      <ul><li>Magdalena (1), Melanie (2), Valentin (3), Rebeccca (4), Christina (5), Martin (6), Mina (7), Lisa (8)</li></ul>
 +
      <ul><li>Dilution of plasmids for Golden Gate: Lisa</li></ul>
 +
  </ul>
 +
 +
<h5 id="dilution">Dilution sheet for Golden Gate Assembly</h5>
 +
<p></p>
 +
<table class="table">
 +
<thead>
 +
<tr>
 +
<th>Sample</th>
 +
                <th>Volume of AD - RO Water [µL]</th>
 +
<th>Volume of plasmid/fragment [µL]</th>
 +
</tr>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td>BB1-C-23-Spy-Cas9(NLS)-49</td>
 +
<td>71,6</td>
 +
                <td>28,4</td>
 +
</tr>
 +
<tr>
 +
<td>BB1-T-34-sce-cyc-50</td>
 +
<td>83,0</td>
 +
                <td>17,0</td>
 +
</tr>
 +
        <tr>
 +
<td>BB1-P-12-syr-pNS-J23101-25</td>
 +
<td>85,1</td>
 +
                <td>14,9</td>
 +
</tr>
 +
        <tr>
 +
<td>BB1-P-12-sce-tdh3-45</td>
 +
<td>82,7</td>
 +
                <td>17,3</td>
 +
</tr>
 +
        <tr>
 +
<td>BB1-T-34-eco-B1001-29</td>
 +
<td>80,4</td>
 +
                <td>19,6</td>
 +
</tr>
 +
        <tr>
 +
<td>BB1-P-12-syn-pm-J23105-23</td>
 +
<td>83,9</td>
 +
                <td>16,1</td>
 +
</tr>
 +
        <tr>
 +
<td>BB1-C-23-syn-GFP-30</td>
 +
<td>82,9</td>
 +
                <td>17,1</td>
 +
</tr>
 +
        <tr>
 +
<td>BB1-P-12-sce-tef1-46</td>
 +
<td>78,0</td>
 +
                <td>22,0</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
 +
<div id="date-7-7">
 +
  <h1>Friday, July 7th.</h1>
 +
<ul>
 +
    <li>Team discussion, Planning and Fundraising</li>
 +
    <li><a href="https://2017.igem.org/Team:BOKU-Vienna/Protocol#interlab" target="_blank">Interlab Challenge</a>: Fluorescein fluorescence standard curve</li>
 +
    <ul><li>Christina</li></ul>
 +
  </ul> 
 +
</div>
 +
 +
<div id="date-7-10">
 +
  <h1>Monday, July 10th.</h1>
 +
  <ul>
 +
    <li>InterLab Study:</li>
 +
      <ul>
 +
        <li>Christina, Felicia</li>
 +
      </ul>
 +
    <li>Dilution of several primers 1:10 (AD-RO Water µL45 , Primer µL5)</li>
 +
      <ul><li>Magdalena</li>
 +
      </ul>
 +
    <li>PCR Gel Preparation</li>
 +
      <ul><li>Mathias</li>
 +
      </ul>
 +
  </ul>
 +
</div>
 +
 +
<div id="date-7-11">
 +
  <h1>Tuesday, July 11th.</h1>
 +
  <ul>
 +
    <li>Planning & Fundraising</li>
 +
    <li>Golden Gate Assembly:</li>
 +
      <ul><li>Flpe (BB1), GFP (BB2) → Transformation (according to protocol) → Plating onto Agar (After Transformation): Selection with Ampicillin (bb1),  Selection with Kanamycin(bb2), stored at 37°C over night
 +
      </li></ul>
 +
    <li>Dilution of several primer</li>
 +
      <ul><table class="table">
 +
<thead>
 +
<tr>
 +
<th>Abbreviation</th>
 +
                <th>Primer</th>
 +
</tr>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td>1_fw</td>
 +
<td>pBAD_fw_FS_1</td>
 +
             
 +
</tr>
 +
<tr>
 +
<td>2_fw</td>
 +
<td>RT_fw_FS2_BpiI</td>
 +
           
 +
</tr>
 +
        <tr>
 +
<td>2_rev</td>
 +
<td>RT_rev_RBS_FS_C_BpiI</td>
 +
             
 +
</tr>
 +
        <tr>
 +
<td>3_fw</td>
 +
<td>FLPe_fw_FS_C_BpiI</td>
 +
</tr>
 +
        <tr>
 +
<td>3_rev</td>
 +
<td>FLPe_rev_FS_3_BpiI</td>
 +
</tr>
 +
        <tr>
 +
<td>4_fw</td>
 +
<td>RepA101ts_fw_FS_23_BsaI</td>
 +
           
 +
</tr>
 +
        <tr>
 +
<td>4_rev</td>
 +
<td>RepA101ts_rev_FS_3_BsaI</td>
 +
</tr>
 +
        <tr>
 +
<td>5_fw</td>
 +
<td>AmpR_Intron_Fw_ohne_FRT_FS_2_BsaI</td>
 +
</tr>
 +
 +
        <tr>
 +
<td>5_rev</td>
 +
<td>AmpR_Intron_rev_ohne_FRT_FS_3_BsaI</td>
 +
</tr>
 +
          <tr>
 +
<td>6_fw</td>
 +
<td>KanR_CripsR_fw_FS_B_BsaI</td>
 +
           
 +
</tr>
 +
        <tr>
 +
<td>6_rev</td>
 +
<td>KanR_CrispR_rev_Fs_E_BsaI</td>
 +
</tr>
 +
        <tr>
 +
<td>7_fw</td>
 +
<td>AmpR_Intron_Fw_FRT_FS_2_BsaI</td>
 +
</tr>
 +
        <tr>
 +
<td>7_rev</td>
 +
<td>AmpR_Intron_rev_FRT_FS_3_BsaI</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
      </ul>
 +
  <li>PCR plasmid fragments</li>
 +
      <ul><li>Josef</li>
 +
      </ul>
 +
        <ul><li>fragment 1: GFP+Primer binding side, fragment 2: GFP + ½ FRT sequence, fragment 3: 1 random sequence
 +
      </li></ul>
 +
  <li>Gel electrophoresis</li>
 +
  </ul>
 +
</div>
 +
 +
<div id="date-7-12">
 +
  <h1>Wednesday, July 12th.</h1>
 +
  <ul>
 +
    <li>Gel PCR for Kanamycin casette</li>
 +
      <ul>
 +
        <li>Mina, Magda</li>
 +
      </ul>
 +
<li>Restriction digest</li>
 +
<li>Gel/PCR DNA Extraction [PROTOCOL]</li>
 +
<li>NEB Golden Gate Assembly Mix [PROTOCOL]</li>
 +
 
 +
  <li>Miniprep [PROTOCOL]</li>
 +
  <ul><li>Lisa</li></ul>
 +
</ul>
 +
</div>
 +
 +
<div id="date-7-3">
 +
  <h1>Monday, July 3rd.</h1>
 +
  <ul>
 +
    <li>Team discussion and overview:</li>
 +
      <ul>
 +
        <li>Christina, Felicia, Josef, Lisa, Magdalena, Mathias, Melanie, Michael, Mina, Rebecca, Valentin, Julian</li>
 +
      </ul>
 +
    <li>Planning of the procedures including primer design</li>
 +
    <li><a href="https://2017.igem.org/Team:BOKU-Vienna/Protocol#lbmedium1" target="_blank">Preparing the growth media and autoclaving</a></li>
 +
      <ul><li>LB medium preparation (4L): Mina, Michael</li></ul>
 +
      <ul><li>Autoclaving: Mina, Rebecca</li></ul>
 +
  </ul>
 +
  <li>InterLab Study</li>
 +
    <ul>
 +
        <li>Felicia, Christina</li>
 +
    </ul>
 +
<li>PCR and gel electrophoresis</li>
 +
    <ul>
 +
        <li>Michael</li>
 +
    </ul>
 +
</div>
 +
 +
<div id="date-7-13">
 +
  <h1>Thursday, July 13th.</h1>
 +
  <ul>
 +
    <li>Miniprep: Flpe + GFP BB2, control digest</li>
 +
      <ul>
 +
        <li>Lisa, Mina, Valentin</li>
 +
      </ul>
 +
    <li>Minis onto LB Kanamycin medium</li>
 +
        <ul><li>Rebecca</li></ul>
 +
   
 +
    <li>Control digest - GFP [PROTOCOL]</li>
 +
      <ul><li>Mina, Lisa</li></ul>
 +
     
 +
    <li>Gel electrophoresis</li>
 +
      <ul><li>Felicia, Mathias</li></ul>
 +
 +
    <li>Golden Gate Assembly:</li>
 +
      <ul><li>Joseph, Felicia, Mina, Christina, Rebecca</li></ul>
 +
  </ul>
 +
</div>
 +
 +
<div id="date-7-14">
 +
  <h1>Friday, July 14th.</h1>
 +
  <ul>
 +
    <li>Transformation from GG-clones in competent cells using LB medium (with Kan or Amp) [PROTOCOL]</li>
 +
      <ul>
 +
        <li>Christina, Joseph, Felicia, Melanie, Rebecca </li>
 +
      </ul>
 +
 +
    <li>Golden Gate Assembly: [PROTOCOL]</li>
 +
      <ul><li>stored at 37°C</li></ul>
 +
      <ul><li>Joseph, Felicia, Mina, Christina, Rebecca, Lisa, Mathias, Valentin</li></ul>
 +
 +
    <li>Gel electrophoresis</li>
 +
      <ul><li>Felicia, Valentin</li></ul>
 +
    <li>Purifiying DNA fragments from elektrophoresis according to given protocol in the HiYield Gel/PCR DNA Extraction Kit[PROTOCOL]</li>
 +
        <ul><li>Felicia, Christina, Valentin, Lisa</li></ul>       
 +
    <li>Measurement of purified DNA</li>
 +
        <ul><li> Julian</li></ul>
 +
  </ul>
 +
</div>
 +
 +
<div id="date-7-17">
 +
  <h1>Monday, July 17th.</h1>
 +
  <ul>
 +
    <li>Miniprep</li>
 +
      <ul>
 +
        <li>control digest</li>
 +
      </ul>
 +
      <ul>
 +
        <li>BB1_10, BB1_11, BB1_12, BB2_2, BB2_3, BB2_4, BB2_5: </li>
 +
      </ul>
 +
      <ul>
 +
        <li>Gel electrophoresis</li>
 +
      </ul>
 +
      <ul>
 +
        <li>Mathias, Mina, Magda, Melanie, Lisa, Rebecca</li>
 +
      </ul>
 +
 +
    <li>Mini: p5IM5 30°C, Kryo</li>
 +
      <ul><li>Rebecca</li></ul>
 +
      <ul><li>Control Transformation</li></ul>
 +
 +
    <li>PCR</li>
 +
      <ul><li>Golden Gate: BB2_6 (Rep A, BB2_7: Cas 9, BB2_8: RT, Flpe, BB2_8 → RT, Flpe coli, BB2_1+BB2_5) + Trafo (on LB AMP)
 +
</li></ul>
 +
    <li>Purifiying DNA fragments from elektrophoresis according to given protocol in the HiYield Gel/PCR DNA Extraction Kit[PROTOCOL]</li>     
 +
    <li>Transformation of Golden Gate products:</li>
 +
        <ul><li>Michi, Julian, Christina, Felicia</li></ul>
 +
 
 +
 +
<li>Kryo for Sequencing</li>
 +
      <ul>
 +
        <li>Kryo: tube with Glycerine + sample</li>
 +
      </ul>
 +
  </ul>
 +
</div>
 +
 +
<div id="date-7-18">
 +
  <h1>Tuesday, July 18th.</h1>
 +
  <ul>
 +
    <li>Load gel for gel electrophoresis</li>
 +
      <ul>
 +
        <li>Melanie</li>
 +
      </ul>
 +
    <li>Media preparation</li>
 +
        <ul>
 +
        <li>Melanie, Valentin</li>
 +
      </ul>
 +
    <li>Colony PCR (11 colonies) [PROTOCOL]</li>
 +
      <ul><li>Magdalena, Felicia, Christina, Mina</li></ul>
 +
 +
    <li>Preparation for Sequencing (cryo)</li>
 +
        <ul>
 +
        <li>Christina</li>
 +
      </ul>
 +
  <li>more colony PCR [PROTOCOL]</li>
 +
        <ul>
 +
        <li>Felicia, Christina, Valentin, Rebecca, Lisa</li>
 +
      </ul>
 +
 +
  <li>PCR Reaction with ThermoCycler</li>
 +
  </ul>
 +
</div>
 +
 +
<div id="date-7-19">
 +
  <h1>Wednesday, July 19th.</h1>
 +
  <ul>
 +
    <li>PCR gel elektrophoresis</li>
 +
      <ul>
 +
        <li>Melanie, Christina, Felicia, Magdalena</li>
 +
      </ul>
 +
    <li>Media preparation (Agar)</li>
 +
        <ul>
 +
        <li>Lisa, Rebecca</li>
 +
      </ul>
 +
    <li>Miniprep [PROTOCOL]</li>
 +
      <ul><li>Christina, Felicia, Valentin, Rebecca</li></ul>
 +
 +
    <li>Preparation for Sequencing</li>
 +
        <ul>
 +
        <li>Christina, Melanie</li>
 +
      </ul>
 +
  <li>Golden Gate [PROTOCOL]</li>
 +
        <ul>
 +
        <li>Mathias, Felicia, Lisa, Rebecca</li>
 +
      </ul>
 +
 +
  <li>Transformation for Golden Gate</li>
 +
      <ul>
 +
        <li>Magdalena, Lisa, Mina, Rebecca, Mathias, Julian, Michael</li>
 +
      </ul>
 +
  </ul>
 +
</div>
 +
 +
 +
<div id="date-7-20">
 +
  <h1>Thursday, July 20th.</h1>
 +
  <ul>
 +
    <li>Colony PCR:</li>
 +
      <ul><li>
 +
      9 samples: BB2_1 A, B, C, D, E, F und BB2_11 A, B, C
 +
      </li>
 +
      </ul>
 +
      <ul>
 +
        <li>Lisa, Christina, Felicia, Rebecca, Lisa, Mina</li>
 +
      </ul>
 +
    <li>Golden Gate repetition (CRISPR: 5alpha, 10B):</li>
 +
        <ul>
 +
        <li>Felicia</li>
 +
      </ul>
 +
    <li>DH10B transformation of Golden Gate products (5alpha, 10B):</li>
 +
      <ul><li>Christina, Felicia</li></ul>
 +
 +
    <li>PCR #5</li>
 +
        <ul>
 +
        <li>Christina</li>
 +
      </ul>
 +
  <li>Gel electrophoresis</li>
 +
        <ul><li>
 +
          with Colony PCR products, Golden Gate products, PCR #5
 +
        </li></ul>
 +
        <ul>
 +
        <li>Felicia, Christina</li>
 +
      </ul>
 +
 +
  <li>DNA purification of PCR #6</li>
 +
      <ul>
 +
        <li>Christina</li>
 +
      </ul>
 +
  <li>Aliquoting Master Mix</li>
 +
      <ul>
 +
        <li>Valentin</li>
 +
      </ul>
 +
  <li>Golden Gate (GFP, BB2_16)</li>
 +
      <ul>
 +
        <li>Valentin, Mathias, Rebecca</li>
 +
      </ul>
 +
  <li>Plating repA colony onto agar</li>
 +
      <ul>
 +
        <li>Lisa, Valentin</li>
 +
      </ul>
 +
  <li>Transformation</li>
 +
      <ul>
 +
        <li>Michael, Julian, Felicia, Christina</li>
 +
      </ul>
 +
  </ul>
 +
</div>
 +
 +
<div id="date-7-21">
 +
  <h1>Friday, July 21st.</h1>
 +
  <ul>
 +
    <li>Minipreparation:</li>
 +
      <ul>
 +
        <li>BB2_11A: Christina</li>
 +
      </ul>
 +
    <li>BB3_5: Yeast in LB Hygromycin</li>
 +
    <li>Colony PCR</li>
 +
      <ul><li>Rebecca, Felicia, Melanie, Christina, Mathias</li></ul>
 +
    <li>Golden Gate PCR - CRISPR</li>
 +
      <ul><li>Christina, Magdalena, Mathias</li></ul>
 +
  </ul>
 +
</div>
 +
 +
<div id="date-7-24">
 +
  <h1>Monday, July 24th.</h1>
 +
  <ul>
 +
    <li>Colony PCR: CRISPR (4x), BB3_5 (6x)</li>
 +
      <ul>
 +
        <li>Lisa, Valentin</li>
 +
      </ul>
 +
    <li>PCR: yeast sgRNA URA3, yeast sgRNA LEV2</li>
 +
      <ul>
 +
      <li>Mathias, Christina, Melanie</li>
 +
      </ul>
 +
    <li>Golden Gate</li>
 +
      <ul><li>BB3_2, BB3_1, BB3_3, BB3_4, BB3_6, BB2_18, BB3_8</li></ul>
 +
      <ul><li>Christina, Mathias</li></ul>
 +
    <li>Golden Gate</li>
 +
        <ul><li>BB1_16, BB1_17</li></ul>
 +
        <ul><li>Mina, Felicia</li></ul>
 +
  </ul>
 +
</div>
 +
 +
</body>
  
  

Revision as of 14:24, 24 July 2017

Notebook

V

Notebook.


Our Notebook is still under construction. You can find our protocol for the methods here: Protocol

Monday, July 3rd.

  • Team discussion and overview:
    • Christina, Felicia, Josef, Lisa, Magdalena, Mathias, Melanie, Michael, Mina, Rebecca, Valentin, Julian
  • Planning of the procedures including primer design
  • Preparing the growth media and autoclaving
    • LB medium preparation (4L): Mina, Michael
    • Autoclaving: Mina, Rebecca

Tuesday, July 4th.

  • Planning and fundraising:
    • Christina, Felicia, Josef, Lisa, Martin, Mathias, Melanie, Michael, Mina, Rebecca, Valentin, Julian
  • Preparation of growth media:
    • 20 plates LB medium + chloramphenicol: Mina, Lisa, Mathias
    • 2 flasks LB agar: Mina, Lisa, Mathias
  • Making cells from straind DH10B chemically competent as a control for the InterLab Study

Wednesday, July 5th.

  • Planning and fundraising:
    • Christina, Felicia, Josef, Lisa, Magdalena, Martin, Mathias, Melanie, Michael, Mina, Rebecca, Julian
  • Aliquoting 1mL LB into Eppendorf tubes
    • Lisa, Josef
  • Transferring Backbones onto agar plates:
    • Julian
  • Interlab challenge - transformation: According to protocol:
      • Lisa, Mathias, Michael, Mina, Rebecca
    • 8 plasmids plated onto agar medium: Positive control (50µL, 200µL), Negative Control (50µL, 200µL), Transformant (20µL, 100µL, 200µL), Test device 1-6 (50µL, 200µL)
    • stored at 35°C

Thursday, July 6th.

  • Team discussion, planning and fundraising
  • Counting colonies of Transformants for the Interlab Challenge:
    • Colonies 200µL: 77, 20µL: 26, 100µL: 43
    Transformants on plate Transformants on plate Transformants on plate
  • Miniprep of 8 plasmids for Golden Gate Standard - according to protocol
    • Magdalena (1), Melanie (2), Valentin (3), Rebeccca (4), Christina (5), Martin (6), Mina (7), Lisa (8)
    • Dilution of plasmids for Golden Gate: Lisa
Dilution sheet for Golden Gate Assembly

Sample Volume of AD - RO Water [µL] Volume of plasmid/fragment [µL]
BB1-C-23-Spy-Cas9(NLS)-49 71,6 28,4
BB1-T-34-sce-cyc-50 83,0 17,0
BB1-P-12-syr-pNS-J23101-25 85,1 14,9
BB1-P-12-sce-tdh3-45 82,7 17,3
BB1-T-34-eco-B1001-29 80,4 19,6
BB1-P-12-syn-pm-J23105-23 83,9 16,1
BB1-C-23-syn-GFP-30 82,9 17,1
BB1-P-12-sce-tef1-46 78,0 22,0

Friday, July 7th.

  • Team discussion, Planning and Fundraising
  • Interlab Challenge: Fluorescein fluorescence standard curve
    • Christina

Monday, July 10th.

  • InterLab Study:
    • Christina, Felicia
  • Dilution of several primers 1:10 (AD-RO Water µL45 , Primer µL5)
    • Magdalena
  • PCR Gel Preparation
    • Mathias

Tuesday, July 11th.

  • Planning & Fundraising
  • Golden Gate Assembly:
    • Flpe (BB1), GFP (BB2) → Transformation (according to protocol) → Plating onto Agar (After Transformation): Selection with Ampicillin (bb1), Selection with Kanamycin(bb2), stored at 37°C over night
  • Dilution of several primer
      • Abbreviation Primer
      1_fw pBAD_fw_FS_1
      2_fw RT_fw_FS2_BpiI
      2_rev RT_rev_RBS_FS_C_BpiI
      3_fw FLPe_fw_FS_C_BpiI
      3_rev FLPe_rev_FS_3_BpiI
      4_fw RepA101ts_fw_FS_23_BsaI
      4_rev RepA101ts_rev_FS_3_BsaI
      5_fw AmpR_Intron_Fw_ohne_FRT_FS_2_BsaI
      5_rev AmpR_Intron_rev_ohne_FRT_FS_3_BsaI
      6_fw KanR_CripsR_fw_FS_B_BsaI
      6_rev KanR_CrispR_rev_Fs_E_BsaI
      7_fw AmpR_Intron_Fw_FRT_FS_2_BsaI
      7_rev AmpR_Intron_rev_FRT_FS_3_BsaI
  • PCR plasmid fragments
    • Josef
    • fragment 1: GFP+Primer binding side, fragment 2: GFP + ½ FRT sequence, fragment 3: 1 random sequence
  • Gel electrophoresis

Wednesday, July 12th.

  • Gel PCR for Kanamycin casette
    • Mina, Magda
  • Restriction digest
  • Gel/PCR DNA Extraction [PROTOCOL]
  • NEB Golden Gate Assembly Mix [PROTOCOL]
  • Miniprep [PROTOCOL]
    • Lisa

Monday, July 3rd.

  • Team discussion and overview:
    • Christina, Felicia, Josef, Lisa, Magdalena, Mathias, Melanie, Michael, Mina, Rebecca, Valentin, Julian
  • Planning of the procedures including primer design
  • Preparing the growth media and autoclaving
    • LB medium preparation (4L): Mina, Michael
    • Autoclaving: Mina, Rebecca
  • InterLab Study
    • Felicia, Christina
  • PCR and gel electrophoresis
    • Michael

    Thursday, July 13th.

    • Miniprep: Flpe + GFP BB2, control digest
      • Lisa, Mina, Valentin
    • Minis onto LB Kanamycin medium
      • Rebecca
    • Control digest - GFP [PROTOCOL]
      • Mina, Lisa
    • Gel electrophoresis
      • Felicia, Mathias
    • Golden Gate Assembly:
      • Joseph, Felicia, Mina, Christina, Rebecca

    Friday, July 14th.

    • Transformation from GG-clones in competent cells using LB medium (with Kan or Amp) [PROTOCOL]
      • Christina, Joseph, Felicia, Melanie, Rebecca
    • Golden Gate Assembly: [PROTOCOL]
      • stored at 37°C
      • Joseph, Felicia, Mina, Christina, Rebecca, Lisa, Mathias, Valentin
    • Gel electrophoresis
      • Felicia, Valentin
    • Purifiying DNA fragments from elektrophoresis according to given protocol in the HiYield Gel/PCR DNA Extraction Kit[PROTOCOL]
      • Felicia, Christina, Valentin, Lisa
    • Measurement of purified DNA
      • Julian

    Monday, July 17th.

    • Miniprep
      • control digest
      • BB1_10, BB1_11, BB1_12, BB2_2, BB2_3, BB2_4, BB2_5:
      • Gel electrophoresis
      • Mathias, Mina, Magda, Melanie, Lisa, Rebecca
    • Mini: p5IM5 30°C, Kryo
      • Rebecca
      • Control Transformation
    • PCR
      • Golden Gate: BB2_6 (Rep A, BB2_7: Cas 9, BB2_8: RT, Flpe, BB2_8 → RT, Flpe coli, BB2_1+BB2_5) + Trafo (on LB AMP)
    • Purifiying DNA fragments from elektrophoresis according to given protocol in the HiYield Gel/PCR DNA Extraction Kit[PROTOCOL]
    • Transformation of Golden Gate products:
      • Michi, Julian, Christina, Felicia
    • Kryo for Sequencing
      • Kryo: tube with Glycerine + sample

    Tuesday, July 18th.

    • Load gel for gel electrophoresis
      • Melanie
    • Media preparation
      • Melanie, Valentin
    • Colony PCR (11 colonies) [PROTOCOL]
      • Magdalena, Felicia, Christina, Mina
    • Preparation for Sequencing (cryo)
      • Christina
    • more colony PCR [PROTOCOL]
      • Felicia, Christina, Valentin, Rebecca, Lisa
    • PCR Reaction with ThermoCycler

    Wednesday, July 19th.

    • PCR gel elektrophoresis
      • Melanie, Christina, Felicia, Magdalena
    • Media preparation (Agar)
      • Lisa, Rebecca
    • Miniprep [PROTOCOL]
      • Christina, Felicia, Valentin, Rebecca
    • Preparation for Sequencing
      • Christina, Melanie
    • Golden Gate [PROTOCOL]
      • Mathias, Felicia, Lisa, Rebecca
    • Transformation for Golden Gate
      • Magdalena, Lisa, Mina, Rebecca, Mathias, Julian, Michael

    Thursday, July 20th.

    • Colony PCR:
      • 9 samples: BB2_1 A, B, C, D, E, F und BB2_11 A, B, C
      • Lisa, Christina, Felicia, Rebecca, Lisa, Mina
    • Golden Gate repetition (CRISPR: 5alpha, 10B):
      • Felicia
    • DH10B transformation of Golden Gate products (5alpha, 10B):
      • Christina, Felicia
    • PCR #5
      • Christina
    • Gel electrophoresis
      • with Colony PCR products, Golden Gate products, PCR #5
      • Felicia, Christina
    • DNA purification of PCR #6
      • Christina
    • Aliquoting Master Mix
      • Valentin
    • Golden Gate (GFP, BB2_16)
      • Valentin, Mathias, Rebecca
    • Plating repA colony onto agar
      • Lisa, Valentin
    • Transformation
      • Michael, Julian, Felicia, Christina

    Friday, July 21st.

    • Minipreparation:
      • BB2_11A: Christina
    • BB3_5: Yeast in LB Hygromycin
    • Colony PCR
      • Rebecca, Felicia, Melanie, Christina, Mathias
    • Golden Gate PCR - CRISPR
      • Christina, Magdalena, Mathias

    Monday, July 24th.

    • Colony PCR: CRISPR (4x), BB3_5 (6x)
      • Lisa, Valentin
    • PCR: yeast sgRNA URA3, yeast sgRNA LEV2
      • Mathias, Christina, Melanie
    • Golden Gate
      • BB3_2, BB3_1, BB3_3, BB3_4, BB3_6, BB2_18, BB3_8
      • Christina, Mathias
    • Golden Gate
      • BB1_16, BB1_17
      • Mina, Felicia