Difference between revisions of "Team:Northwestern/08 04"

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   <article>
 
   <article>
 
     <h1>Thursday, August 4<sup>th</sup></h3>
 
     <h1>Thursday, August 4<sup>th</sup></h3>
−
     <h2>Agenda:</h2>
+
     <h2>Tasks:</h2>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Jordan</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li> Transformed the Golden Gate projects:
 +
            <ul>
 +
              <li>Left on ice for ten seconds, then all reactions were pipetted into cells within one minute</li>
 +
              <li>Added 600 uL SOC (realized I left some tubes off ice for a sec while I was adding media but that hopefully shouldn’t be a problem)</li>
 +
              <li>Plated all 650 uL</li>
 +
            </ul>
 +
          </li>
 +
          <li>Transformed GFP/MCherry storage vectors:
 +
            <ul>
 +
              <li>Used 600 uL of SOC compared to usual 450</li>
 +
            </ul>
 +
          </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Michelle</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>GFP Gel extraction</li>
 +
          <li> Made more femtomole dilutions
 +
            <ul>
 +
              <li>Tet backbone: made 25 μL more
 +
                <ul>
 +
                  <li>11.84 μL of 27.4 ng/μL stock to 12.5 μL</li>
 +
                  <li>4.35 μL of 37.3 ng/μL stock to 6.25 μL</li>
 +
                  <li>4.41 μL of 36.8 ng/μL stock to 6.25 μL</li>
 +
                </ul>
 +
              </li>
 +
              <li>GFP1: made 10 μL more
 +
                <ul>
 +
                  <li>2.74 μL of 72.2 ng/μL stock to 10 μL</li>
 +
                </ul>
 +
              </li>
 +
              <li>GFP2: made 10 μL more
 +
                <ul>
 +
                  <li>2.30 μL of 86.0 ng/μL stock to 10 μL</li>
 +
                </ul>
 +
              </li>
 +
            </ul>
 +
          </li>
 +
          <li>PCR Tet backbone to linearize for GFP in case this golden gate doesn’t work
 +
            <ul>
 +
              <li>2 x 50 μL reactions
 +
                <ul>
 +
                  <li>20 μL nfH<sub>2</sub>O</li>
 +
                  <li>1 μL DMSO</li>
 +
                  <li>2 μL Tet backbone template (from miniprep)</li>
 +
                  <li>1 μL 10μM forward primer</li>
 +
                  <li>1 μL 10μM reverse primer</li>
 +
                  <li>25 μL OneTaq Master Mix</li>
 +
                </ul>
 +
              </li>
 +
              <li>1 negative control (no template)
 +
                <ul>
 +
                  <li>22 μL nfH<sub>2</sub>O</li>
 +
                  <li>1 μL DMSO</li>
 +
                  <li>1 μL 10μM forward primer</li>
 +
                  <li>1 μL 10μM reverse primer</li>
 +
                  <li>25 μL OneTaq Master Mix</li>
 +
                </ul>
 +
              </li>
 +
            </ul>
 +
          </li>
 +
          <li>Poured a gel to run the Tet linearization for GFP (Michelle)
 +
            <ul>
 +
              <li>~20–30 mL of 1% agarose</li>
 +
              <li>2 μL SybrGreen</li>
 +
            </ul>
 +
          </li>
 +
          <li>DpnI digested the Tet linearized for GFP PCR product (Michelle)
 +
            <ul>
 +
              <li>0.5 μL of DpnI added to each 50 μL PCR tube</li>
 +
              <li>Did not digest the negative control tube</li>
 +
            </ul>
 +
          </li>
 +
          <li>Plated ligation products of the Golden Gate parts into storage vectors</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
<div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Sara</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Made cam plates
 +
          <ul><li>275 mL LB</li>
 +
          <li>4.5 g bacto agar</li>
 +
          <li>Autoclave, then 275 uL Cam</li></ul></li>
 +
          <li>Emailed Quentin about getting 6X purple loading dye from the Jewett lab</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Shu</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li> Retransform the Gibson products in the freezer
 +
            <ul>
 +
              <li>3uL 1:2 product (Shu made)</li>
 +
              <li>3uL 1:3 product (Shu made)</li>
 +
              <li>3uL product (Jordan made)</li>
 +
              <li>1uL positive control from competent cell kit (in J04450- pSB1C3)</li>
 +
              <li>1uL positive control from Gibson kit</li>
 +
              <li>1uL water</li>
 +
              <li>200µL of SOC (as per Kelly’s protocol)</li>
 +
              <li>Plated the entire tube </li>
 +
            </ul>
 +
          </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 
</article>
 
</article>
 
<footer id="nav">
 
<footer id="nav">

Revision as of 17:18, 15 October 2016

Notebook

Thursday, August 4th

Tasks:

Jordan

  • Transformed the Golden Gate projects:
    • Left on ice for ten seconds, then all reactions were pipetted into cells within one minute
    • Added 600 uL SOC (realized I left some tubes off ice for a sec while I was adding media but that hopefully shouldn’t be a problem)
    • Plated all 650 uL
  • Transformed GFP/MCherry storage vectors:
    • Used 600 uL of SOC compared to usual 450

Michelle

  • GFP Gel extraction
  • Made more femtomole dilutions
    • Tet backbone: made 25 μL more
      • 11.84 μL of 27.4 ng/μL stock to 12.5 μL
      • 4.35 μL of 37.3 ng/μL stock to 6.25 μL
      • 4.41 μL of 36.8 ng/μL stock to 6.25 μL
    • GFP1: made 10 μL more
      • 2.74 μL of 72.2 ng/μL stock to 10 μL
    • GFP2: made 10 μL more
      • 2.30 μL of 86.0 ng/μL stock to 10 μL
  • PCR Tet backbone to linearize for GFP in case this golden gate doesn’t work
    • 2 x 50 μL reactions
      • 20 μL nfH2O
      • 1 μL DMSO
      • 2 μL Tet backbone template (from miniprep)
      • 1 μL 10μM forward primer
      • 1 μL 10μM reverse primer
      • 25 μL OneTaq Master Mix
    • 1 negative control (no template)
      • 22 μL nfH2O
      • 1 μL DMSO
      • 1 μL 10μM forward primer
      • 1 μL 10μM reverse primer
      • 25 μL OneTaq Master Mix
  • Poured a gel to run the Tet linearization for GFP (Michelle)
    • ~20–30 mL of 1% agarose
    • 2 μL SybrGreen
  • DpnI digested the Tet linearized for GFP PCR product (Michelle)
    • 0.5 μL of DpnI added to each 50 μL PCR tube
    • Did not digest the negative control tube
  • Plated ligation products of the Golden Gate parts into storage vectors

Sara

  • Made cam plates
    • 275 mL LB
    • 4.5 g bacto agar
    • Autoclave, then 275 uL Cam
  • Emailed Quentin about getting 6X purple loading dye from the Jewett lab

Shu

  • Retransform the Gibson products in the freezer
    • 3uL 1:2 product (Shu made)
    • 3uL 1:3 product (Shu made)
    • 3uL product (Jordan made)
    • 1uL positive control from competent cell kit (in J04450- pSB1C3)
    • 1uL positive control from Gibson kit
    • 1uL water
    • 200µL of SOC (as per Kelly’s protocol)
    • Plated the entire tube