Difference between revisions of "Team:Northwestern/08 30"

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   <article>
 
   <article>
 
     <h1>Tuesday, August 30<sup>th</sup></h3>
 
     <h1>Tuesday, August 30<sup>th</sup></h3>
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<h2>Tasks:</h2>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Jordan</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Started cytosolic GFP cultures for tomorrow</li>
 +
          <li>Interviewed Dr. Postelnick</li>
 +
          <li>Worked on travel grant</li>
 +
          <li>Read instructions for millipore filters </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Michelle</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Ran western blot</li>
 +
          <ul>
 +
            <li>We successfully expressed Cas9
 +
              <div class="row">
 +
                <div class="col-xs-8 col-sm-12"><img src="https://static.igem.org/mediawiki/2016/b/bd/T--Northwestern--08_30_1.png" width="1498" height="838" alt=""/></div>
 +
              </div>
 +
              <div class="row">
 +
                <div class="col-xs-8 col-sm-12"><img src="https://static.igem.org/mediawiki/2016/6/67/T--Northwestern--08_30_2.png" width="1228" height="687" alt=""/></div>
 +
              </div>
 +
            </li>
 +
            <li>There are no bands in lanes 2-4 because Thush didn’t induce the promoter but Ben offered to run another Western after inducing</li>
 +
            <li>It was a good control</li>
 +
            <li>Cas9 seems to appear between the 97 and 191 kDa bands, but closer to the 97 kDa band, which is expected (~114 kDa is what we’re going for)</li>
 +
            <li>Each of the “Cas9 Assembled” lanes have a lot more bands than expected, and this could be attributed to truncated protein expression and cleavage sites</li>
 +
            <li>Kelly can give us cells we can run a western on as a control, to see if the extra bands on lanes 5-7 are attributed to noise </li>
 +
          </ul>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Sam</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Interviewed Dr. Postelnick</li>
 +
          <li>Emailed Dr. Tullman-Ercek</li>
 +
          <li>Cited antibiotics pamphlet for adults</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Tasfia</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>DpnI digest of Cas9-Lrz-SS PCR products from 8.29.16</li>
 +
          <li>Golden Gate PCR for sfGFP and mCherry (nine reactions, one 50-uL reaction for each) </li>
 +
          <ul>
 +
            <li>sfGFP taken from 8.23.16 miniprep</li>
 +
            <li>mCherry taken from 7.35.16 miniprep </li>
 +
            <li>Amount of template used:
 +
              <div class="row">
 +
                <div class="col-xs-8 col-sm-6"><img src="https://static.igem.org/mediawiki/2016/4/49/T--Northwestern--08_30_3.png" width="1228" height="687" alt=""/></div>
 +
              </div>
 +
            </li>
 +
            <li>Conditions: 95&#176;C (5:00) | 95&#176;C (0:07), 54&#176;C (0:10), 72&#176;C (0:43) | 72&#176;C (2:00)</li>
 +
          </ul>
 +
          <li>Ran gels on Tet-Lrz-GFP (8.29.16), Cas9-Lrz-SS (8.29.16), and all the GFP/mCherry for GG PCR products (8.30.16)</li>
 +
          <ul>
 +
            <li>Used SybrSafe from Jewett lab (bands were very faint on all gels)</li>
 +
            <li>Recommended: Use ~3 uL SybrSafe on small 10-well gels (with SybrGreen you can get away with ~1 uL)</li>
 +
            <li>These gels used ~3 uL on the 14-well gels </li>
 +
          </ul>
 +
          <div class="row">
 +
            <div class="col-xs-8 col-sm-6"><img src="https://static.igem.org/mediawiki/2016/9/9a/T--Northwestern--08_30_5.png" width="1228" height="687" alt=""/>
 +
              <p>2kB band is very faint</p>
 +
            </div>
 +
            <div class="col-xs-8 col-sm-6"><img src="https://static.igem.org/mediawiki/2016/1/11/T--Northwestern--08_30_6.png" width="1228" height="687" alt=""/></div>
 +
          </div>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Tyler</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Redesigned primers for restriction digestion of Cas9 into PSB1C3</li>
 +
          <li>Looked into reusing primers for sequencing of gRNA</li>
 +
          <li>Transformation test with Cam plates</li>
 +
          <ul>
 +
            <li>Cam and Tet plate test results</li>
 +
            <ul>
 +
              <li>Tet plates prepared with Kelly’s LB grew exponentially more colonies than tet plates prepared with our LB on August 8th</li>
 +
              <li>gRNA Gibson product grew one colony</li>
 +
              <li>Nothing grew on plates prepared with our LB, plates prepared with Kelly’s LB, or Kelly’s already-prepared plates from Leonard lab</li>
 +
              <li>There’s probably something wrong with our positive control plasmid for Cam</li>
 +
            </ul>
 +
          </ul>
 +
          <li>Drafted e-mail to Tullman-Ercek w/Sam </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
  </article>
 +
 
 
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<footer id="nav">
 
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Revision as of 01:22, 16 October 2016

Notebook

Tuesday, August 30th

Tasks:

Jordan

  • Started cytosolic GFP cultures for tomorrow
  • Interviewed Dr. Postelnick
  • Worked on travel grant
  • Read instructions for millipore filters

Michelle

  • Ran western blot
    • We successfully expressed Cas9
    • There are no bands in lanes 2-4 because Thush didn’t induce the promoter but Ben offered to run another Western after inducing
    • It was a good control
    • Cas9 seems to appear between the 97 and 191 kDa bands, but closer to the 97 kDa band, which is expected (~114 kDa is what we’re going for)
    • Each of the “Cas9 Assembled” lanes have a lot more bands than expected, and this could be attributed to truncated protein expression and cleavage sites
    • Kelly can give us cells we can run a western on as a control, to see if the extra bands on lanes 5-7 are attributed to noise

Sam

  • Interviewed Dr. Postelnick
  • Emailed Dr. Tullman-Ercek
  • Cited antibiotics pamphlet for adults

Tasfia

  • DpnI digest of Cas9-Lrz-SS PCR products from 8.29.16
  • Golden Gate PCR for sfGFP and mCherry (nine reactions, one 50-uL reaction for each)
    • sfGFP taken from 8.23.16 miniprep
    • mCherry taken from 7.35.16 miniprep
    • Amount of template used:
    • Conditions: 95°C (5:00) | 95°C (0:07), 54°C (0:10), 72°C (0:43) | 72°C (2:00)
  • Ran gels on Tet-Lrz-GFP (8.29.16), Cas9-Lrz-SS (8.29.16), and all the GFP/mCherry for GG PCR products (8.30.16)
    • Used SybrSafe from Jewett lab (bands were very faint on all gels)
    • Recommended: Use ~3 uL SybrSafe on small 10-well gels (with SybrGreen you can get away with ~1 uL)
    • These gels used ~3 uL on the 14-well gels

    2kB band is very faint

Tyler

  • Redesigned primers for restriction digestion of Cas9 into PSB1C3
  • Looked into reusing primers for sequencing of gRNA
  • Transformation test with Cam plates
    • Cam and Tet plate test results
      • Tet plates prepared with Kelly’s LB grew exponentially more colonies than tet plates prepared with our LB on August 8th
      • gRNA Gibson product grew one colony
      • Nothing grew on plates prepared with our LB, plates prepared with Kelly’s LB, or Kelly’s already-prepared plates from Leonard lab
      • There’s probably something wrong with our positive control plasmid for Cam
  • Drafted e-mail to Tullman-Ercek w/Sam