Difference between revisions of "Team:Northwestern/07 27"

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   <article>
 
   <article>
 
     <h1>Wednesday, July 27<sup>th</sup></h3>
 
     <h1>Wednesday, July 27<sup>th</sup></h3>
−
    <h2>Agenda:</h2>
+
  <h2>Agenda:</h2>
−
 
+
    <ul>
 +
      <li> Pour 2 gels for GFP and mCherry</li>
 +
      <li>PCR linearization of the Tet-Cas9 backbone in preparation for insertion of ClyA or INP/signaling sequences</li>
 +
      <li>PCR Cleanup of GG gels</li>
 +
    </ul>
 +
    <h2>Tasks:</h2>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Jordan</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Designed ClyA sequencing primers</li>
 +
          <li>Ran gel extraction/cleanup on mCherry’s (Shu did GFPs), followed kit procedure, used 30 ul water not elution buffer, added more buffer up to about 500 ul</li>
 +
          <li>Looked into more fractionation procedures Ligation </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Michelle</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li> • Made glycerol stocks of A1–A3, A5, B1–B3, B5 Cas9 cultures
 +
<ul>
 +
  <li>500 μL overnight culture</li>
 +
  <li>500 μL 50% glycerol in dH<sub>2</sub>O</li>
 +
  <li>In microcentrifuge tube stored in -80°C in Mordacq’s lab, in box marked 2016 iGEM Glycerol Stocks</li>
 +
</ul>
 +
          </li>
 +
          <li>Researched Cas9 expression &amp; purification protocols</li>
 +
          <li>Cas9–INP and Cas9–ClyA Gibson math</li>
 +
          <li>Made 5mL cultures of Cas9 from glycerol stocks with Sara
 +
<ul>
 +
  <li>50 μL Cam in 5 mL LB</li>
 +
  <li>Cultures A3, A5, B2</li>
 +
            </ul>
 +
          </li>
 +
          <li>Ran mCherry &amp; sfGFP PCR
 +
          <ul><li>0.5 μL of 10 μM forward primer</li>
 +
          <li>0.5 μL of 10 μM reverse primer</li>
 +
          <li>1 μL template (sfGFP or mCherry miniprep)</li>
 +
          <li>12.5 μL of OneTaq Master Mix</li>
 +
          <li>10.5 μL nuclease-free water</li>
 +
          <li>95&#176;C (2:00) | 95&#176;C (0:07), &nbsp;51&#176;C (0:10), &nbsp;72&#176;C (0:43) | 72&#176;C (5:00)</li>
 +
          <li>25 μL reaction volume, 25 cycles </li></ul></li>
 +
          <li>Aliquoted 100 mL of LB for autoclaving to grow up Cas9 tomorrow</li>
 +
          <li>Aliquoted LB into smaller bottles for autoclaving tomorrow </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Paul</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Looked up Cas9 expression procedure</li>
 +
          <li>Ran gel on mCherry and GFP PCR products</li>
 +
          <li>Helped out with various experiments</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 Tet-Cas9 backbone with Tyler
 +
          <ul><li>Used DNA from B2 and A5 overnight cultures</li></ul></li>
 +
          <li>Made 5mL cultures of Cas9 from glycerol stocks with Michelle
 +
          <ul><li>50 μL Cam in 5 mL LB</li>
 +
          <li>Cultures A3, A5, B2 </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>Gel extracted sfGFP for Golden Gate assembly</li>
 +
          <li>Grew mRFP culture</li>
 +
          <li>Looked over SS sequencing primers</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Tasfia</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Made glycerol stocks with Michelle</li>
 +
          <li>PCR Cleanup of GFP/mCherry</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
</article>
 
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<footer id="nav">
 
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     </footer>
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Revision as of 06:10, 15 October 2016

Notebook

Wednesday, July 27th

Agenda:

  • Pour 2 gels for GFP and mCherry
  • PCR linearization of the Tet-Cas9 backbone in preparation for insertion of ClyA or INP/signaling sequences
  • PCR Cleanup of GG gels

Tasks:

Jordan

  • Designed ClyA sequencing primers
  • Ran gel extraction/cleanup on mCherry’s (Shu did GFPs), followed kit procedure, used 30 ul water not elution buffer, added more buffer up to about 500 ul
  • Looked into more fractionation procedures Ligation

Michelle

  • • Made glycerol stocks of A1–A3, A5, B1–B3, B5 Cas9 cultures
    • 500 μL overnight culture
    • 500 μL 50% glycerol in dH2O
    • In microcentrifuge tube stored in -80°C in Mordacq’s lab, in box marked 2016 iGEM Glycerol Stocks
  • Researched Cas9 expression & purification protocols
  • Cas9–INP and Cas9–ClyA Gibson math
  • Made 5mL cultures of Cas9 from glycerol stocks with Sara
    • 50 μL Cam in 5 mL LB
    • Cultures A3, A5, B2
  • Ran mCherry & sfGFP PCR
    • 0.5 μL of 10 μM forward primer
    • 0.5 μL of 10 μM reverse primer
    • 1 μL template (sfGFP or mCherry miniprep)
    • 12.5 μL of OneTaq Master Mix
    • 10.5 μL nuclease-free water
    • 95°C (2:00) | 95°C (0:07),  51°C (0:10),  72°C (0:43) | 72°C (5:00)
    • 25 μL reaction volume, 25 cycles
  • Aliquoted 100 mL of LB for autoclaving to grow up Cas9 tomorrow
  • Aliquoted LB into smaller bottles for autoclaving tomorrow

Paul

  • Looked up Cas9 expression procedure
  • Ran gel on mCherry and GFP PCR products
  • Helped out with various experiments

Sara

  • PCR of Tet-Cas9 backbone with Tyler
    • Used DNA from B2 and A5 overnight cultures
  • Made 5mL cultures of Cas9 from glycerol stocks with Michelle
    • 50 μL Cam in 5 mL LB
    • Cultures A3, A5, B2

Shu

  • Gel extracted sfGFP for Golden Gate assembly
  • Grew mRFP culture
  • Looked over SS sequencing primers

Tasfia

  • Made glycerol stocks with Michelle
  • PCR Cleanup of GFP/mCherry