Difference between revisions of "Team:Northwestern/09 14"

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   <article>
 
   <article>
 
     <h1>Wednesday, September 14<sup>th</sup></h3>
 
     <h1>Wednesday, September 14<sup>th</sup></h3>
−
     <h2>Agenda:</h2>
+
     <h2>Tasks:</h2>
−
 
+
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Jordan</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Wrote “Experiments” doc for the website</li>
 +
          <li>Transformed Gibson products</li>
 +
          <ul><li>YcdO, NapA, ClyA, INP, AmiA, FhuD, negative control with only backbone- 3uL, positive control with 50 pg of RFP- 1 uL</li>
 +
          <li>All on Cam</li>
 +
          <li>Plated 500 uL of 50 uL cells + 950 uL SOC</li>
 +
          <li>Incubation= 1 hr 15, heat shock 30 seconds </li></ul>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
<div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Sam</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Wrote Auburn Gresham scaffold</li>
 +
          <li>Looked over presentation, sent to Emma</li>
 +
          <li>Emailed Dr. DeLisa</li>
 +
          <li>Edited presentation</li>
 +
          <li>Updated OneNote</li>
 +
          <li>To do: redesign survey </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Sara</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Miniprepped Ben’s cell line, pET28a: 23 ng/uL, 260/280: 2.01, 260/230: 2.14</li>
 +
          <li>Made a glycerol stock of these cells</li>
 +
          <li>Made glycerol stock of Cas9-Dsba (1)</li>
 +
          <li>Made glycerol stock of gRNA (4)</li>
 +
          <li>Finished transforming the golden gate transformations </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Tasfia</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>PCR Cleanup of INP with homology and Cas9 for restriction digest</li>
 +
          <li>Gel screen on Tet-Lrz-GFP from PCR troubleshooting </li>
 +
          <div class="row">
 +
            <div class="col-xs-6"><img src="https://static.igem.org/mediawiki/2016/1/17/T--Northwestern--09_14_1.png" width="245" height="349" alt=""/></div>
 +
            <div class="col-xs-6"><img src="https://static.igem.org/mediawiki/2016/7/79/T--Northwestern--09_14_2.png" width="245" height="349" alt=""/></div>
 +
          </div>
 +
          <li>DpnI good Tet-Lrz-GFP PCR products (Lanes 3-5) from 09.13.16</li>
 +
          <li>PCR Cleanup of Tet-Lrz-GFP after DpnI
 +
            <div class="row">
 +
              <div class="col-xs-6"><img src="https://static.igem.org/mediawiki/2016/9/9e/T--Northwestern--09_14_3.png" width="245" height="349" alt=""/></div>
 +
            </div>
 +
          </li>
 +
          <li>Gibson Assemblies (Shu set up the reactions, Tyler made the calculations, and Sara/I reviewed the calculations #GoTeam)</li>
 +
          <ul>
 +
            <li>Cas9-NapA </li>
 +
            <ul>
 +
              <li>0.32 μL NapA with PCR homology insert</li>
 +
              <li>0.98 μL Cas9-Lrz-SS backbone</li>
 +
              <li>3.70 μL nuclease-free water</li>
 +
              <li>5.00 μL Gibson Master Mix</li>
 +
            </ul>
 +
            <li>Cas9-AmiA</li>
 +
            <ul>
 +
              <li>0.16 μL AmiA with PCR homology insert</li>
 +
              <li>0.98 μL Cas9-Lrz-SS backbone</li>
 +
              <li>3.86 μL nuclease-free water</li>
 +
              <li>5.00 μL Gibson Master Mix</li>
 +
            </ul>
 +
            <li>Cas9-YcdO </li>
 +
            <ul>
 +
              <li>0.16 μL YcdO with PCR homology insert </li>
 +
              <li>0.98 μL Cas9-Lrz-SS backbone </li>
 +
              <li>3.86 μL nuclease-free water </li>
 +
              <li>5.00 μL Gibson Master Mix </li>
 +
            </ul>
 +
            <li>Cas9-FhuD </li>
 +
            <ul>
 +
              <li>0.20 μL FhuD with PCR homoloy insert </li>
 +
              <li>0.98 μL Cas9-Lrz-SS backbone </li>
 +
              <li>3.82 μL nuclease-free water </li>
 +
              <li>5.00 μL Gibson Master Mix </li>
 +
            </ul>
 +
            <li>Cas9-ClyA </li>
 +
            <ul>
 +
              <li>1.63 μL ClyA gBlock insert</li>
 +
              <li>0.98 μL Cas9-Lrz-ClyA backbone </li>
 +
              <li>2.39 μL nuclease-free water </li>
 +
              <li>5.00 μL Gibson Master Mix </li>
 +
            </ul>
 +
            <li>Cas9-INP </li>
 +
            <ul>
 +
              <li>0.65 μL diluted INP (1:9 DNA-to-water dilution) with PCR homology insert </li>
 +
              <li>0.98 μL Cas9-Lrz-SS backbone </li>
 +
              <li>3.37 μL nuclease-free water </li>
 +
              <li>5.00 μL Gibson Master Mix </li>
 +
            </ul>
 +
            <li>Negative Control </li>
 +
            <ul>
 +
              <li>0.98 μL Cas9-Lrz-ClyA backbone </li>
 +
              <li>4.02 μL nuclease-free water </li>
 +
              <li>5.00 μL Gibson Master Mix </li>
 +
            </ul>
 +
            <li>Reactions are running for an hour at 50&#176;C</li>
 +
          </ul>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Tyler</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Reviewed sequencing reactions</li>
 +
          <ul>
 +
            <li>For Dsba
 +
              2,4,7,8,9, A, and B have suboptimal sequencing results, but still look to be usable</li>
 +
            <ul>
 +
              <li>USE 1,3,5,6,10</li>
 +
            </ul>
 +
            <li>For gRNA, gRNA 6 and 7 had suboptimal results and were thrown away</li>
 +
            <ul>
 +
              <li>USE 1,2,3,4,5,8,9,10</li>
 +
            </ul>
 +
          </ul>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
  </article>
 
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Latest revision as of 15:35, 18 October 2016

Notebook

Wednesday, September 14th

Tasks:

Jordan

  • Wrote “Experiments” doc for the website
  • Transformed Gibson products
    • YcdO, NapA, ClyA, INP, AmiA, FhuD, negative control with only backbone- 3uL, positive control with 50 pg of RFP- 1 uL
    • All on Cam
    • Plated 500 uL of 50 uL cells + 950 uL SOC
    • Incubation= 1 hr 15, heat shock 30 seconds

Sam

  • Wrote Auburn Gresham scaffold
  • Looked over presentation, sent to Emma
  • Emailed Dr. DeLisa
  • Edited presentation
  • Updated OneNote
  • To do: redesign survey

Sara

  • Miniprepped Ben’s cell line, pET28a: 23 ng/uL, 260/280: 2.01, 260/230: 2.14
  • Made a glycerol stock of these cells
  • Made glycerol stock of Cas9-Dsba (1)
  • Made glycerol stock of gRNA (4)
  • Finished transforming the golden gate transformations

Tasfia

  • PCR Cleanup of INP with homology and Cas9 for restriction digest
  • Gel screen on Tet-Lrz-GFP from PCR troubleshooting
  • DpnI good Tet-Lrz-GFP PCR products (Lanes 3-5) from 09.13.16
  • PCR Cleanup of Tet-Lrz-GFP after DpnI
  • Gibson Assemblies (Shu set up the reactions, Tyler made the calculations, and Sara/I reviewed the calculations #GoTeam)
    • Cas9-NapA
      • 0.32 μL NapA with PCR homology insert
      • 0.98 μL Cas9-Lrz-SS backbone
      • 3.70 μL nuclease-free water
      • 5.00 μL Gibson Master Mix
    • Cas9-AmiA
      • 0.16 μL AmiA with PCR homology insert
      • 0.98 μL Cas9-Lrz-SS backbone
      • 3.86 μL nuclease-free water
      • 5.00 μL Gibson Master Mix
    • Cas9-YcdO
      • 0.16 μL YcdO with PCR homology insert
      • 0.98 μL Cas9-Lrz-SS backbone
      • 3.86 μL nuclease-free water
      • 5.00 μL Gibson Master Mix
    • Cas9-FhuD
      • 0.20 μL FhuD with PCR homoloy insert
      • 0.98 μL Cas9-Lrz-SS backbone
      • 3.82 μL nuclease-free water
      • 5.00 μL Gibson Master Mix
    • Cas9-ClyA
      • 1.63 μL ClyA gBlock insert
      • 0.98 μL Cas9-Lrz-ClyA backbone
      • 2.39 μL nuclease-free water
      • 5.00 μL Gibson Master Mix
    • Cas9-INP
      • 0.65 μL diluted INP (1:9 DNA-to-water dilution) with PCR homology insert
      • 0.98 μL Cas9-Lrz-SS backbone
      • 3.37 μL nuclease-free water
      • 5.00 μL Gibson Master Mix
    • Negative Control
      • 0.98 μL Cas9-Lrz-ClyA backbone
      • 4.02 μL nuclease-free water
      • 5.00 μL Gibson Master Mix
    • Reactions are running for an hour at 50°C

Tyler

  • Reviewed sequencing reactions
    • For Dsba 2,4,7,8,9, A, and B have suboptimal sequencing results, but still look to be usable
      • USE 1,3,5,6,10
    • For gRNA, gRNA 6 and 7 had suboptimal results and were thrown away
      • USE 1,2,3,4,5,8,9,10