Difference between revisions of "Team:Northwestern/07 28"

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   <article>
 
   <article>
 
     <h1>Thursday, July 28<sup>th</sup></h3>
 
     <h1>Thursday, July 28<sup>th</sup></h3>
−
     <h2>Agenda:</h2>
+
     <h2>Sequencing Results:</h2>
−
 
+
    <p>Cas9 Gibson cultures A1, A2, A3, A5, B1, B2, B3, B5: Only the BBprefix_FWD primer gave us clean results, showing that the Cas9 was not successfully inserted into the linearized backbone. We realized that we used circular DNA in the Gibson assembly.</p>
 +
    <h2>Tasks:</h2>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Jordan</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Transformed the GFP and mCherry ligations
 +
          <ul><li>Ran all 9 tests, did a backbone-only ligation control, a plate with no DNA, and a control from the competent cell test (on Cam)</li>
 +
          <li>Transformed 4 uL of plasmid DNA each, 1 uL for no DNA and comp cell controls</li>
 +
          <li>Long ice incubation step because water bath wasn’t ready yet, ~45 minutes</li></ul></li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Michelle</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li> Autoclaved
 +
          <ul><li>LB media from 7/26/16 into smaller aliquot bottles</li>
 +
          <li>Three beveled Erlenmeyers of 100 mL of LB for Cas9 expression cultures</li>
 +
          <li>Pipette tips</li>
 +
          <li>Eppendorf tubes </li></ul></li>
 +
         
 +
          <li>Took a fridge inventory</li>
 +
          <li>Vortexed all crashed out T4 Ligase Buffers</li>
 +
         
 +
         
 +
          <li>Reran the linearizing Tet backbone PCR
 +
<ul><li>0.5 μL of 10 μM forward primer</li>
 +
              <li>0.5 μL of 10 μM reverse primer</li>
 +
              <li>1.0 μL Tet in pSB1C3 template (~2100 bp)</li>
 +
              <li>12.5 μL 2X Q5 Master Mix</li>
 +
              <li>10.5 μL nuclease-free water</li>
 +
              <li>95&#176;C (2:00) | 95&#176;C (0:07), &nbsp;63&#176;C (0:10), &nbsp;72&#176;C (2:06) | 72&#176;C (5:00)</li>
 +
              <li>25 μL reaction volume, 25 cycles </li></ul></li>
 +
          <li>Made T4 Ligase Buffer Aliquots (20 μL)</li>
 +
          <li>Ran gel on Tet backbone PCR results
 +
          <ul>
 +
          <li>Lane 1: 2 μL of 2-log Purple ladder + 6 μL of NEB Blue 6X loading dye</li>
 +
          <li>Lane 3: 25 μL PCR reaction + 5 μL NEB Blue 6X loading dye</li>
 +
          </ul>
 +
          </li>
 +
          <li>RTW-PCR linearized Tet backbone to troubleshoot Master Mixes and Taq polymerase—Taq failure</li>
 +
          <div class="row">
 +
            <div class="col-sm-6"><img src="https://static.igem.org/mediawiki/2016/8/8c/T--Northwestern--07_28_1.jpg" width="987" height="968" alt=""/>
 +
              <p><strong>Figure 1:</strong> PCR results. Lane 1: 2log ladder. Lane 2: No insert control. Lane 4: Master Mix, 0.5uL primers. Lane 6: Separate components, 0.5uL primers. Lane 8: Master Mix, 1.25uL primers. Lane 10: Separate components, 1.25uL primers</p>
 +
            </div>
 +
          </div>
 +
        </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 for Michelle</li>
 +
          <li>Analyzed sequencing</li>
 +
          <li>Ran colony PCR
 +
            </li>
 +
          <li>12.5 uL OneTaq MM</li>
 +
          <li>0.5 uL BB Vf Primer</li>
 +
          <li>0.5 uL BB Vr Primer</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Shu</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Miniprep of mRFP in pSB1T3
 +
          <ul><li>Made two stocks</li>
 +
          <li>#1: 70.3ng/uL, 260/280:1.93, 260/230:1.93</li>
 +
          <li>#2: 63.8ng/uL, 260/280:1.93, 260/230:1.95</li></ul></li>
 +
          <li>Ran a gel for GFP PCR product</li>
 +
          <li>Gel extraction with Thush</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Tasfia</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Ran gel and gel extraction for GFP and mCherry</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
</article>
 
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Latest revision as of 15:28, 18 October 2016

Notebook

Thursday, July 28th

Sequencing Results:

Cas9 Gibson cultures A1, A2, A3, A5, B1, B2, B3, B5: Only the BBprefix_FWD primer gave us clean results, showing that the Cas9 was not successfully inserted into the linearized backbone. We realized that we used circular DNA in the Gibson assembly.

Tasks:

Jordan

  • Transformed the GFP and mCherry ligations
    • Ran all 9 tests, did a backbone-only ligation control, a plate with no DNA, and a control from the competent cell test (on Cam)
    • Transformed 4 uL of plasmid DNA each, 1 uL for no DNA and comp cell controls
    • Long ice incubation step because water bath wasn’t ready yet, ~45 minutes

Michelle

  • Autoclaved
    • LB media from 7/26/16 into smaller aliquot bottles
    • Three beveled Erlenmeyers of 100 mL of LB for Cas9 expression cultures
    • Pipette tips
    • Eppendorf tubes
  • Took a fridge inventory
  • Vortexed all crashed out T4 Ligase Buffers
  • Reran the linearizing Tet backbone PCR
    • 0.5 μL of 10 μM forward primer
    • 0.5 μL of 10 μM reverse primer
    • 1.0 μL Tet in pSB1C3 template (~2100 bp)
    • 12.5 μL 2X Q5 Master Mix
    • 10.5 μL nuclease-free water
    • 95°C (2:00) | 95°C (0:07),  63°C (0:10),  72°C (2:06) | 72°C (5:00)
    • 25 μL reaction volume, 25 cycles
  • Made T4 Ligase Buffer Aliquots (20 μL)
  • Ran gel on Tet backbone PCR results
    • Lane 1: 2 μL of 2-log Purple ladder + 6 μL of NEB Blue 6X loading dye
    • Lane 3: 25 μL PCR reaction + 5 μL NEB Blue 6X loading dye
  • RTW-PCR linearized Tet backbone to troubleshoot Master Mixes and Taq polymerase—Taq failure

    • Figure 1: PCR results. Lane 1: 2log ladder. Lane 2: No insert control. Lane 4: Master Mix, 0.5uL primers. Lane 6: Separate components, 0.5uL primers. Lane 8: Master Mix, 1.25uL primers. Lane 10: Separate components, 1.25uL primers

Sara

  • Poured a gel for Michelle
  • Analyzed sequencing
  • Ran colony PCR
  • 12.5 uL OneTaq MM
  • 0.5 uL BB Vf Primer
  • 0.5 uL BB Vr Primer

Shu

  • Miniprep of mRFP in pSB1T3
    • Made two stocks
    • #1: 70.3ng/uL, 260/280:1.93, 260/230:1.93
    • #2: 63.8ng/uL, 260/280:1.93, 260/230:1.95
  • Ran a gel for GFP PCR product
  • Gel extraction with Thush

Tasfia

  • Ran gel and gel extraction for GFP and mCherry