Difference between revisions of "Team:Northwestern/09 20"

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     <h1>Tuesday, September 20<sup>th</sup></h3>
 
     <h1>Tuesday, September 20<sup>th</sup></h3>
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    <h2>Agenda:</h2>
+
  <h2>Tasks:</h2>
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+
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Jordan</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Helped make competent Delisa cells</li>
 +
          <li>Ligated Cas9 and pSB1C3 restriction digests</li>
 +
          <ul>
 +
            <li>25 ng pSB1C3 (2ul)</li>
 +
            <li>37 ng Cas9 insert (1.86 uL)</li>
 +
            <li>1 ul T4 ligase buffer</li>
 +
            <li>0.5 ul T4 ligase</li>
 +
            <li>4.64 ul water</li>
 +
            <li>1:1 insert:vector</li>
 +
            <li>16&#176;for 30 minutes, heat kill 80&#176;C 20 minutes </li>
 +
          </ul>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Sam</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Electroporated 4 lines of cells with Paul</li>
 +
          <li>Skipped the negative control because the Jewett lab only had 4 electroporation cuvettes </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Sara</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Poured a gel to screen the clyA-PET28a PCR</li>
 +
          <ul>
 +
            <li>3 uL sybersafe</li>
 +
            <li>5 uL product, 1 uL dye</li>
 +
            <li>6 uL ladder, 3 uL dye</li>
 +
            <li>The gel showed that the PCR did not work</li>
 +
          </ul>
 +
          <li>Made more 1% agarose (400 mL)</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Tasfia</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Made competent cells out of DeLisa strain (with Paul and Jordan)</li>
 +
          <li>Transformed ligation product (Cas9 into pSB1C3 iGEM backbone for part submission), 3 μL DNA</li>
 +
          <li>Transformed pSB1C3 competent cell test plasmid to examine efficiency of DeLisa competent cells</li>
 +
          <li>Picked more colonies of gRNA+Cas9 cotransformation, and with existing overnight cultures made 2.5-mL aliquots to give cultures more air in the tubes </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Tyler</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Gibson Reactions</li>
 +
          <ul>
 +
            <li>Cas9 </li>
 +
            <ul>
 +
              <li>0.74 µL backbone (1:2 dilution) </li>
 +
              <li>1.15 µL insert (1:5 dilution) </li>
 +
              <li>3.11 µL water </li>
 +
              <li>5 µL mix </li>
 +
            </ul>
 +
            <li>SS-Cas9 DsbA </li>
 +
            <ul>
 +
              <li>0.74 µL backbone </li>
 +
              <li>0.75 µL insert (1:5 dilution) </li>
 +
              <li>3.51 µL water </li>
 +
              <li>5 µL mix</li>
 +
            </ul>
 +
            <li>SS-Cas9 Ycdo</li>
 +
            <ul>
 +
              <li>0.74 µL backbone </li>
 +
              <li>0.95 µL insert (1:5 dilution) </li>
 +
              <li>3.31 µL water </li>
 +
              <li>5 µL mix Backbone </li>
 +
            </ul>
 +
            <li>(-) control </li>
 +
            <ul>
 +
              <li>0.74 µL backbone </li>
 +
              <li>4.26 µL water </li>
 +
              <li>5µL mix</li>
 +
            </ul>
 +
          </ul>
 +
        </ul>
 +
      </div>
 +
    </div>
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  </article>
 
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<script type="text/javascript" src="https://2016.igem.org/Team:Northwestern/libraries/jquery?action=raw&ctype=text/javascript"></script>
 
<script type="text/javascript" src="https://2016.igem.org/Team:Northwestern/libraries/jquery?action=raw&ctype=text/javascript"></script>
 
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Revision as of 04:46, 16 October 2016

Notebook

Tuesday, September 20th

Tasks:

Jordan

  • Helped make competent Delisa cells
  • Ligated Cas9 and pSB1C3 restriction digests
    • 25 ng pSB1C3 (2ul)
    • 37 ng Cas9 insert (1.86 uL)
    • 1 ul T4 ligase buffer
    • 0.5 ul T4 ligase
    • 4.64 ul water
    • 1:1 insert:vector
    • 16°for 30 minutes, heat kill 80°C 20 minutes

Sam

  • Electroporated 4 lines of cells with Paul
  • Skipped the negative control because the Jewett lab only had 4 electroporation cuvettes

Sara

  • Poured a gel to screen the clyA-PET28a PCR
    • 3 uL sybersafe
    • 5 uL product, 1 uL dye
    • 6 uL ladder, 3 uL dye
    • The gel showed that the PCR did not work
  • Made more 1% agarose (400 mL)

Tasfia

  • Made competent cells out of DeLisa strain (with Paul and Jordan)
  • Transformed ligation product (Cas9 into pSB1C3 iGEM backbone for part submission), 3 μL DNA
  • Transformed pSB1C3 competent cell test plasmid to examine efficiency of DeLisa competent cells
  • Picked more colonies of gRNA+Cas9 cotransformation, and with existing overnight cultures made 2.5-mL aliquots to give cultures more air in the tubes

Tyler

  • Gibson Reactions
    • Cas9
      • 0.74 µL backbone (1:2 dilution)
      • 1.15 µL insert (1:5 dilution)
      • 3.11 µL water
      • 5 µL mix
    • SS-Cas9 DsbA
      • 0.74 µL backbone
      • 0.75 µL insert (1:5 dilution)
      • 3.51 µL water
      • 5 µL mix
    • SS-Cas9 Ycdo
      • 0.74 µL backbone
      • 0.95 µL insert (1:5 dilution)
      • 3.31 µL water
      • 5 µL mix Backbone
    • (-) control
      • 0.74 µL backbone
      • 4.26 µL water
      • 5µL mix