Difference between revisions of "Team:Northwestern/08 23"

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     <h1>Tuesday, August 23<sup>rd</sup></h3>
 
     <h1>Tuesday, August 23<sup>rd</sup></h3>
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    <h2>Agenda:</h2>
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  <h2>Tasks:</h2>
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    <div class="row">
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      <div class="col-sm-2">
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        <p>Jordan</p>
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      </div>
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      <div class="col-sm-10">
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        <ul>
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          <li>Did more research into periplasm fractioning
 +
          <ul><li>In order to see if the protocol works, need JC8031 with ClyA culture, a JC8031 DeLisa cell line control (same cells, no GFP) and a cytoplasmic GFP control (if there is GFP in the fraction, full cell lysis happened and that’s bad)</li>
 +
          <li>Plan:
 +
          <ul><li>Grow up overnight culture of GFP device from interlab kit </li>
 +
          <li>Grow all three cultures in 25 mL LB cultures with Cam until OD=0.5 per DeLisa paper</li>
 +
          <li>Induce JC8301 cells with .2% arabinose (weight/final volume)</li>
 +
          <li>Grow for 6 hours at 37</li>
 +
          <li>Use cold osmotic shock protocol from Biefeld iGEM team on the drive, BUT use ice cold water instead of cell fraction buffer 2 because Kelly says Triton and SDS can interfere with protein function</li>
 +
          <li>Measure fractions on plate reader, use wavelength from interlab study</li></ul></li>
 +
          <li>Need: to make cell fraction buffer (need Tris) autoclave media?, grow up cytoplasm GFP, did Paul make some arabinose?</li></ul></li>
 +
          <li>Made some 3M sodium acetate solution to try ethanol precipitation—Kelly says it’s worth a try—see Paul for final pH</li>
 +
          <li>Ethanol precipitation <a href="http://openwetware.org/wiki/Ethanol_precipitation_of_nucleic_acids">protocol</a></li>
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        </ul>
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      </div>
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    </div>
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 +
 
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<div class="row">
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      <div class="col-sm-2">
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        <p>Paul</p>
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      </div>
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      <div class="col-sm-10">
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        <ul>
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          <li>Looked into Cas9 expression cell lines with Kelly</li>
 +
          <li>Cas9 should be constitutively expressed in any line without TetR repressor (so our cell line)</li>
 +
          <li>Gibson assemble gRNA into mRFP reporter plasmid pSB1T3
 +
            <ul>
 +
              <li>Used Bradley’s Clone Kit Excel calculator
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                <div class="row">
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                  <div class="col-sm-12"><img src="https://static.igem.org/mediawiki/2016/5/59/T--Northwestern--08_23_1.jpg" width="838" height="245" alt=""/></div>
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                </div>
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              </li>
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              <li>Pos. Gibson Control</li>
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              <li>Neg Gibson Control (no insert)</li>
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              <li>10 uL rxns:
 +
                <ul>
 +
                  <li>4.15 uL nfH20</li>
 +
                  <li>0.41 uL 63 ng/uL mRFP backbone</li>
 +
                  <li>0.45 uL 10 ng/uL gRNA gBlock insert</li>
 +
                  <li>5 uL Gibson Mastermix </li>
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                </ul>
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              </li>
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            </ul>
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          </li>
 +
          <li>Note for 8/24: L-arabinose 0.2% final w/v for induction of ClyA GFP </li>
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        </ul>
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      </div>
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Revision as of 22:48, 15 October 2016

Notebook

Tuesday, August 23rd

Tasks:

Jordan

  • Did more research into periplasm fractioning
    • In order to see if the protocol works, need JC8031 with ClyA culture, a JC8031 DeLisa cell line control (same cells, no GFP) and a cytoplasmic GFP control (if there is GFP in the fraction, full cell lysis happened and that’s bad)
    • Plan:
      • Grow up overnight culture of GFP device from interlab kit
      • Grow all three cultures in 25 mL LB cultures with Cam until OD=0.5 per DeLisa paper
      • Induce JC8301 cells with .2% arabinose (weight/final volume)
      • Grow for 6 hours at 37
      • Use cold osmotic shock protocol from Biefeld iGEM team on the drive, BUT use ice cold water instead of cell fraction buffer 2 because Kelly says Triton and SDS can interfere with protein function
      • Measure fractions on plate reader, use wavelength from interlab study
    • Need: to make cell fraction buffer (need Tris) autoclave media?, grow up cytoplasm GFP, did Paul make some arabinose?
  • Made some 3M sodium acetate solution to try ethanol precipitation—Kelly says it’s worth a try—see Paul for final pH
  • Ethanol precipitation protocol

Paul

  • Looked into Cas9 expression cell lines with Kelly
  • Cas9 should be constitutively expressed in any line without TetR repressor (so our cell line)
  • Gibson assemble gRNA into mRFP reporter plasmid pSB1T3
    • Used Bradley’s Clone Kit Excel calculator
    • Pos. Gibson Control
    • Neg Gibson Control (no insert)
    • 10 uL rxns:
      • 4.15 uL nfH20
      • 0.41 uL 63 ng/uL mRFP backbone
      • 0.45 uL 10 ng/uL gRNA gBlock insert
      • 5 uL Gibson Mastermix
  • Note for 8/24: L-arabinose 0.2% final w/v for induction of ClyA GFP