Difference between revisions of "Team:Northwestern/07 25"

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     <h1>Monday, July 25<sup>th</sup></h3>
 
     <h1>Monday, July 25<sup>th</sup></h3>
 
     <h2>Agenda:</h2>
 
     <h2>Agenda:</h2>
−
 
+
    <ul>
 +
      <li> Check the GG storage vector</li>
 +
      <li>Freezer inventory</li>
 +
      <li>Figure out sequencing of Cas9</li>
 +
      <li>Review constructs</li>
 +
      <li>Human practices</li>
 +
      <li>Find protocols for next steps</li>
 +
      <li>Design sequencing primers for the GFP construct</li>
 +
      <li>Ligate GFP and mCherry parts into the storage vector</li>
 +
      <li>Look into Weinberg and Provost travel grants</li>
 +
    </ul>
 +
    <h2>Tasks:</h2>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Jordan</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Discovered pSB1A3 contains BsaI site, not suitable for GFP storage vector</li>
 +
          <li>Looked for periplasmic lysis protocols</li>
 +
          <li>Scheduled call with Dr. Persell</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Michelle</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Freezer inventory &amp; organization</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Paul</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Figured out sequencing submission process</li>
 +
          <li>Planned ligation</li>
 +
          <li>Planned gRNA plasmid</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Sam</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Emailed profs regarding Eligo Bioscience</li>
 +
          <li>Prepped questions for Dr. Persell</li>
 +
          <li>Website work</li>
 +
          <li>Troubleshot gel imager</li>
 +
          <li>Proposed that gels should be ran at a lower voltage for longer duration in order to prevent “band smiling”</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Sara</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>PCR of GFP and mCherry
 +
          <ul>
 +
            <li>Miniprep of overnight BB part cultures:
 +
                <ul>
 +
                <li> GFP concentration: 39.7 ng/uL, 260/280: 2.02, 260/230: 1.44</li>
 +
                    <li>mCherry concentration: 38 ng/uL, 260/280: 1.9, 260/230: 1.01</li>
 +
                </ul>
 +
                </li>
 +
                <li>Procedure:
 +
                <ul>
 +
                <li> 2.5 uL 10X PCR buffer</li>
 +
<li>0.5 uL 10 mM DNTPs</li>
 +
<li>0.5 uL of forward primer at 10 mM</li>
 +
<li>0.5 uL of reverse primer at 10 mM</li>
 +
<li>0.5 uL template (Shu's miniprep GFP and mCherry products)</li>
 +
<li>0.25 Taq polymerase</li>
 +
<li>20.25 uL H20</li>
 +
                </ul>
 +
                </li>
 +
                <li>PCR settings:
 +
                <ul>
 +
                <li> Denature: 95 C, 7 s.</li>
 +
<li>Anneal: 51 C, 43 s.</li>
 +
<li>Elongate 72 C, 2 m.</li>
 +
                </ul>
 +
                </li>
 +
            </ul>
 +
          </li>
 +
          <li>Made 400 mL 1% agarose with Shu</li>
 +
          <li>Poured 2 gels</li>
 +
          <li>Ran a gel of the PCR products
 +
          <ul>
 +
            <li>GFP3 and 4 did not have bands in the correct place, so we reran PCR on them and on GFP 2, which had a lower concentration and 260/230 ratio than the rest</li>
 +
<li>We plan to troubleshoot by changing the voltage we’re running at and using newly diluted TAE</li>
 +
            </ul>
 +
          </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Shu</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>PCR of GFP and mCherry
 +
            <ul>
 +
              <li>Miniprep of overnight BB part cultures:
 +
                <ul>
 +
                  <li> GFP concentration: 39.7 ng/uL, 260/280: 2.02, 260/230: 1.44</li>
 +
                  <li>mCherry concentration: 38 ng/uL, 260/280: 1.9, 260/230: 1.01</li>
 +
                </ul>
 +
              </li>
 +
              <li>Procedure:
 +
                <ul>
 +
                  <li> 2.5 uL 10X PCR buffer</li>
 +
                  <li>0.5 uL 10 mM DNTPs</li>
 +
                  <li>0.5 uL of forward primer at 10 mM</li>
 +
                  <li>0.5 uL of reverse primer at 10 mM</li>
 +
                  <li>0.5 uL template (Shu's miniprep GFP and mCherry products)</li>
 +
                  <li>0.25 Taq polymerase</li>
 +
                  <li>20.25 uL H20</li>
 +
                </ul>
 +
              </li>
 +
              <li>PCR settings:
 +
                <ul>
 +
                  <li> Denature: 95 C, 7 s.</li>
 +
                  <li>Anneal: 51 C, 43 s.</li>
 +
                  <li>Elongate 72 C, 2 m.</li>
 +
                </ul>
 +
              </li>
 +
            </ul>
 +
          </li>
 +
          <li>Made 400 mL 1% agarose with Sara</li>
 +
          <li>Poured 2 gels</li>
 +
          <li>Ran a gel of the PCR products
 +
            <ul>
 +
              <li>GFP3 and 4 did not have bands in the correct place, so we reran PCR on them and on GFP 2, which had a lower concentration and 260/230 ratio than the rest</li>
 +
              <li>We plan to troubleshoot by changing the voltage we’re running at and using newly diluted TAE</li>
 +
            </ul>
 +
          </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Tasfia</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>PCR on miniprepped INP</li>
 +
          <li>Ran gel on INP PCR product</li>
 +
          <li>Web content </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Tyler</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Designed gRNA template w/homology to mRFP plasmid, mRFP insert with correct GG sticky ends, reverse primer</li>
 +
          <li>Sponsors comm (Bio-rad)</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
  </article>
 
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Latest revision as of 15:27, 18 October 2016

Notebook

Monday, July 25th

Agenda:

  • Check the GG storage vector
  • Freezer inventory
  • Figure out sequencing of Cas9
  • Review constructs
  • Human practices
  • Find protocols for next steps
  • Design sequencing primers for the GFP construct
  • Ligate GFP and mCherry parts into the storage vector
  • Look into Weinberg and Provost travel grants

Tasks:

Jordan

  • Discovered pSB1A3 contains BsaI site, not suitable for GFP storage vector
  • Looked for periplasmic lysis protocols
  • Scheduled call with Dr. Persell

Michelle

  • Freezer inventory & organization

Paul

  • Figured out sequencing submission process
  • Planned ligation
  • Planned gRNA plasmid

Sam

  • Emailed profs regarding Eligo Bioscience
  • Prepped questions for Dr. Persell
  • Website work
  • Troubleshot gel imager
  • Proposed that gels should be ran at a lower voltage for longer duration in order to prevent “band smiling”

Sara

  • PCR of GFP and mCherry
    • Miniprep of overnight BB part cultures:
      • GFP concentration: 39.7 ng/uL, 260/280: 2.02, 260/230: 1.44
      • mCherry concentration: 38 ng/uL, 260/280: 1.9, 260/230: 1.01
    • Procedure:
      • 2.5 uL 10X PCR buffer
      • 0.5 uL 10 mM DNTPs
      • 0.5 uL of forward primer at 10 mM
      • 0.5 uL of reverse primer at 10 mM
      • 0.5 uL template (Shu's miniprep GFP and mCherry products)
      • 0.25 Taq polymerase
      • 20.25 uL H20
    • PCR settings:
      • Denature: 95 C, 7 s.
      • Anneal: 51 C, 43 s.
      • Elongate 72 C, 2 m.
  • Made 400 mL 1% agarose with Shu
  • Poured 2 gels
  • Ran a gel of the PCR products
    • GFP3 and 4 did not have bands in the correct place, so we reran PCR on them and on GFP 2, which had a lower concentration and 260/230 ratio than the rest
    • We plan to troubleshoot by changing the voltage we’re running at and using newly diluted TAE

Shu

  • PCR of GFP and mCherry
    • Miniprep of overnight BB part cultures:
      • GFP concentration: 39.7 ng/uL, 260/280: 2.02, 260/230: 1.44
      • mCherry concentration: 38 ng/uL, 260/280: 1.9, 260/230: 1.01
    • Procedure:
      • 2.5 uL 10X PCR buffer
      • 0.5 uL 10 mM DNTPs
      • 0.5 uL of forward primer at 10 mM
      • 0.5 uL of reverse primer at 10 mM
      • 0.5 uL template (Shu's miniprep GFP and mCherry products)
      • 0.25 Taq polymerase
      • 20.25 uL H20
    • PCR settings:
      • Denature: 95 C, 7 s.
      • Anneal: 51 C, 43 s.
      • Elongate 72 C, 2 m.
  • Made 400 mL 1% agarose with Sara
  • Poured 2 gels
  • Ran a gel of the PCR products
    • GFP3 and 4 did not have bands in the correct place, so we reran PCR on them and on GFP 2, which had a lower concentration and 260/230 ratio than the rest
    • We plan to troubleshoot by changing the voltage we’re running at and using newly diluted TAE

Tasfia

  • PCR on miniprepped INP
  • Ran gel on INP PCR product
  • Web content

Tyler

  • Designed gRNA template w/homology to mRFP plasmid, mRFP insert with correct GG sticky ends, reverse primer
  • Sponsors comm (Bio-rad)