Difference between revisions of "Team:Northwestern/09 06"

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     <h1>Tuesday, September 6<sup>th</sup></h3>
 
     <h1>Tuesday, September 6<sup>th</sup></h3>
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     <h2>Agenda:</h2>
+
     <h2>Tasks:</h2>
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+
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Jordan</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <div class="row">
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            <div class="col-xs-10"><img src="https://static.igem.org/mediawiki/2016/1/11/T--Northwestern--09_03_1.jpg" width="1600" height="1200" alt=""/>
 +
              <p><strong>Figure 1:</strong> Troubleshooting summary</p>
 +
            </div>
 +
          </div>
 +
          <li>Ran periplasm fraction protocol on Cas9 culture in TetR</li>
 +
          <ul>
 +
            <li>Centrifuged 25 mL suspension for 20 minutes at 4000 rpm and 4C</li>
 +
            <li>Discarded supernatant</li>
 +
            <li>Resuspended in 6.25 mL of ice-cold sucrose cell fractioning buffer 1</li>
 +
            <li>Incubated for 20 minutes, shook periodically</li>
 +
            <li>Centrifuged for 30 minutes at 4000 rpm and 4 degrees</li>
 +
            <li>Discarded supernatant</li>
 +
            <li>Resuspended in 1.56 mL ice cold buffer 2 with no detergents, transferred to eppendorf tube</li>
 +
            <li>Incubated for 20 minutes on ice</li>
 +
            <li>Centrifuged for 15 minutes at 11000 rpm on tabletop centrifuge</li>
 +
            <li>Saved supernatant, in fridge</li>
 +
          </ul>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Michelle</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Gel extracted Cas9 for SS and Cas9 for iGEM</li>
 +
          <ul>
 +
            <li>Nanodrop results:</li>
 +
            <ul>
 +
              <li>Cas9 lnrz for SS: 9.0 ng/uL, 260/280: 1.8, 260/230: 0.44</li>
 +
              <li>Cas9 res.dig. for iGEM: 8.8 ng/uL, 260/280: 1.85, 260/230: 0.43 </li>
 +
            </ul>
 +
          </ul>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Paul</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Got sequencing back for gRNA assembled construct</li>
 +
          <ul>
 +
            <li>Results aligned under “Assembled Parts&gt;pSB1T3…+gRNA”</li>
 +
            <li>Few point mutations on mRFP and Terminator-likely not too bad</li>
 +
          </ul>
 +
          <li>Gibson assembly of YcdO (10 uL: *6:1 insert:bb, 1% DMSO, 4 hrs @50 C*)</li>
 +
          <ul>
 +
            <li>4.4 uL of Cas9 lnrz for SS (from 9/1/16 PCR, gel extracted 9/6/16- 9 ng/uL)</li>
 +
            <li>0.5 uL of YcdO (from PCR products, 38 ng/uL)</li>
 +
            <li>0.1 uL DMSO</li>
 +
            <li>5 uL Gibson Assembly HiFi MasterMix</li>
 +
          </ul>
 +
          <li>Met w/ Joe (w/ Tyler) about modeling</li>
 +
          <ul>
 +
            <li>Detect potential off target cleavage sites of our gRNA in the gut microbiome</li>
 +
          </ul>
 +
          <li>Restriction digest linear pSB1C3 and linear Cas9 (EcoRI, SpeI) </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Sam</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Sent Emma Nechamkin emails</li>
 +
          <ul>
 +
            <li>Grew up two cultures (25 mL to start, ~22 mL now exist)</li>
 +
            <li>Both JC8031</li>
 +
            <ul>
 +
              <li>One has pClyA-GFP-His6 Cam</li>
 +
              <ul>
 +
                <li>Induced with 290 uL arabinose at about .5 absorbance (actual absorbance labelled)</li>
 +
              </ul>
 +
              <li>One is normal</li>
 +
              <ul>
 +
                <li>No antibiotic</li>
 +
              </ul>
 +
            </ul>
 +
            <li>Overnight in the 37&#176;C shaking incubator</li>
 +
          </ul>
 +
          <li>Found and watched antibiotics and CRISPR videos</li>
 +
          <li>Started adding graphics to pamphlet</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Tyler</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Mathematical modeling meeting w/Joe</li>
 +
          <li>Transformation of ligated products + Gibson SS (Ycdo)</li>
 +
          <ul><li>3µL Ligated Cas9 Product</li>
 +
          <li>5µL Gibson SS Product</li>
 +
          <li>1µL Transformation control (50pg mRFP control)</li>
 +
          <li>2µL Gibson (+) control </li></ul>
 +
        </ul>
 +
      </div>
 +
    </div>
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Revision as of 02:13, 16 October 2016

Notebook

Tuesday, September 6th

Tasks:

Jordan

    Figure 1: Troubleshooting summary

  • Ran periplasm fraction protocol on Cas9 culture in TetR
    • Centrifuged 25 mL suspension for 20 minutes at 4000 rpm and 4C
    • Discarded supernatant
    • Resuspended in 6.25 mL of ice-cold sucrose cell fractioning buffer 1
    • Incubated for 20 minutes, shook periodically
    • Centrifuged for 30 minutes at 4000 rpm and 4 degrees
    • Discarded supernatant
    • Resuspended in 1.56 mL ice cold buffer 2 with no detergents, transferred to eppendorf tube
    • Incubated for 20 minutes on ice
    • Centrifuged for 15 minutes at 11000 rpm on tabletop centrifuge
    • Saved supernatant, in fridge

Michelle

  • Gel extracted Cas9 for SS and Cas9 for iGEM
    • Nanodrop results:
      • Cas9 lnrz for SS: 9.0 ng/uL, 260/280: 1.8, 260/230: 0.44
      • Cas9 res.dig. for iGEM: 8.8 ng/uL, 260/280: 1.85, 260/230: 0.43

Paul

  • Got sequencing back for gRNA assembled construct
    • Results aligned under “Assembled Parts>pSB1T3…+gRNA”
    • Few point mutations on mRFP and Terminator-likely not too bad
  • Gibson assembly of YcdO (10 uL: *6:1 insert:bb, 1% DMSO, 4 hrs @50 C*)
    • 4.4 uL of Cas9 lnrz for SS (from 9/1/16 PCR, gel extracted 9/6/16- 9 ng/uL)
    • 0.5 uL of YcdO (from PCR products, 38 ng/uL)
    • 0.1 uL DMSO
    • 5 uL Gibson Assembly HiFi MasterMix
  • Met w/ Joe (w/ Tyler) about modeling
    • Detect potential off target cleavage sites of our gRNA in the gut microbiome
  • Restriction digest linear pSB1C3 and linear Cas9 (EcoRI, SpeI)

Sam

  • Sent Emma Nechamkin emails
    • Grew up two cultures (25 mL to start, ~22 mL now exist)
    • Both JC8031
      • One has pClyA-GFP-His6 Cam
        • Induced with 290 uL arabinose at about .5 absorbance (actual absorbance labelled)
      • One is normal
        • No antibiotic
    • Overnight in the 37°C shaking incubator
  • Found and watched antibiotics and CRISPR videos
  • Started adding graphics to pamphlet

Tyler

  • Mathematical modeling meeting w/Joe
  • Transformation of ligated products + Gibson SS (Ycdo)
    • 3µL Ligated Cas9 Product
    • 5µL Gibson SS Product
    • 1µL Transformation control (50pg mRFP control)
    • 2µL Gibson (+) control