Difference between revisions of "Team:UCLA/Notebook"

(Undo revision 176135 by Yashess (talk))
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   <div class="row" id="pcage-team-journal">
 
   <div class="row" id="pcage-team-journal">
  
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<object data="https://static.igem.org/mediawiki/2016/0/04/Protein_cage_journal.pdf" type="application/pdf" width="1080" height="1000" internalinstanceid="3" title=""></object>
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<iframe src="https://docs.google.com/document/d/1Xsvg65ESjD9rNhsqqEwlT9L-RR-hdX4qVW2nUbJysJE/pub?embedded=true" width="100%" style="height: 100vh;"></iframe>
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<div class="week-entry">
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  <div class="row week-header" id="pcage-week-1-header">
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    <div class="columns small-2 week-number">
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      <span>Week</span>
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      <p>1</p>
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    </div>
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    <div class="columns small-10">
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      <h4>Week 1</h4>
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    </div>
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  </div>
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  <div class="row week-content" id="pcage-week-1-content">
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    <div class="row day-entry">
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      <div class="columns small-2 small-offset-1 day-date">
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        <span>June</span>
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        <p>13</p>
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      </div>
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      <div class="columns small-9">
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        <h5>Plate Making and Transformation of O3-33 WT</h5>
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        <p><p dir="ltr">
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              Goals for today: We have a vector of O333 from Neil King contained in a pET vector, the goal for today is to transform this vector, grow it tomorrow and
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              PCR it for a large number of copies to work with and creation of a biobrick. Primers will need to be designed including the T7 promoter to terminator, with
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              iGEM prefix and suffix.
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          </p>
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          <br/>
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          <p dir="ltr">
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              Made plates
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          </p>
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          <ul>
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              <li dir="ltr">
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                  <p dir="ltr">
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                      12.5g LB
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                  </p>
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              </li>
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              <li dir="ltr">
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                  <p dir="ltr">
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                      7.5 Bacto Agar
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                  </p>
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              </li>
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              <li dir="ltr">
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                  <p dir="ltr">
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                      500mL dH2O
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                  </p>
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              </li>
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              <li dir="ltr">
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                  <p dir="ltr">
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                      500uL Kanomycin (50mg/mL)
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                  </p>
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              </li>
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          </ul>
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          <br/>
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          <ul>
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              <li dir="ltr">
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                  <p dir="ltr">
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                      20 minutes autoclave
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                  </p>
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              </li>
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              <li dir="ltr">
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                  <p dir="ltr">
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                      4 hours total time to solidify
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                  </p>
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              </li>
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          </ul>
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          <p dir="ltr">
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              =&gt; 32 plates total
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          </p>
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          <br/>
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          <p dir="ltr">
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              Transformation
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          </p>
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          <ul>
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              <li dir="ltr">
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                  <p dir="ltr">
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                      30uL iGEM electrocompetent cells
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                  </p>
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              </li>
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              <li dir="ltr">
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−
                  <p dir="ltr">
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                      1uL O333 vector 1:100 dilution
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                  </p>
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              </li>
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              <li dir="ltr">
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                  <p dir="ltr">
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                      970uL SOC save
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                  </p>
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              </li>
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          </ul>
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          <br/>
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          <ul>
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              <li dir="ltr">
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                  <p dir="ltr">
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                      45 minutes incubation @ 800rpm/37C
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                  </p>
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              </li>
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              <li dir="ltr">
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                  <p dir="ltr">
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                      1 and 1:10 dilution plating
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                  </p>
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              </li>
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          </ul>
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          <p dir="ltr">
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              =&gt; 3 transformations total
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          </p>
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          <p dir="ltr">
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              =&gt; arc time: 5.5s, 5.6s, 5.6s
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          </p>
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          <br/>
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          <p dir="ltr">
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              **Plates in 37C inoculation from 6:40pm
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          </p>
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          <br/>
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          <p dir="ltr">
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              Conclusions: Transformations had a consistent arc time. Tomorrow morning plates will be checked for colonies and grown.
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          </p></p>
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      </div>
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    </div>
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    <div class="row day-entry">
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      <div class="columns small-2 small-offset-1 day-date">
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        <span>June</span>
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        <p>14</p>
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      </div>
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      <div class="columns small-9">
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        <h5>Transformed O3-33 WT</h5>
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          <p dir="ltr">
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−
              Recap from previous day: No colonies were shown from any of the transformations. Competent cells may have been of low quality (relatively long arc times).
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          </p>
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          <p dir="ltr">
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              <strong>
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                  <br/>
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              </strong>
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          </p>
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          <p dir="ltr">
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−
              Goals: Today, commercial electro-competent cells are going to be transformed, and different concentrations of DNA will be tested for transformation as
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              well.
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          </p>
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          <p dir="ltr">
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              <strong>
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−
                  <br/>
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              </strong>
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          </p>
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          <p dir="ltr">
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              Transformation:
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          </p>
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          <ul>
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−
              <li dir="ltr">
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−
                  <p dir="ltr">
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−
                      3x 25uL commercial electrocompetent cells
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                  </p>
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              </li>
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              <li dir="ltr">
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−
                  <p dir="ltr">
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−
                      1uL of 1:5, 1:50, 1:100 serial dilutions of plasmid from Neil King
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                  </p>
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              </li>
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−
              <li dir="ltr">
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−
                  <p dir="ltr">
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−
                      970uL SOC save
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                  </p>
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              </li>
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          </ul>
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          <p dir="ltr">
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              <strong>
+
−
                  <br/>
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−
              </strong>
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          </p>
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          <ul>
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              <li dir="ltr">
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−
                  <p dir="ltr">
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−
                      60 minutes save (800rpm @ 37C), then additional 50 minutes at 37C incubator (no shaking) as I was waiting for plating beads to be autoclaved and
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                      cooled
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                  </p>
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              </li>
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              <li dir="ltr">
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−
                  <p dir="ltr">
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                      1 and 1:10 dilution platings
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                  </p>
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              </li>
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          </ul>
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          <p dir="ltr">
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              =&gt; 1:5 DNA: 5.1s arc time
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          </p>
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          <p dir="ltr">
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              =&gt; 1:50 DNA: 5.0s arc time
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          </p>
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          <p dir="ltr">
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              =&gt; 1:100 DNA: 4.8s arc time
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          </p>
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          <p dir="ltr">
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              <strong>
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−
                  <br/>
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              </strong>
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          </p>
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          <p dir="ltr">
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              **Plates in 37C incubation from 4:10pm
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          </p>
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          <p dir="ltr">
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              <br/>
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              Conclusions: Same as yesterday, though today arc times were slightly better. Unfortunately, my timing was off for plating because I was waiting to
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              autoclave new beads (thanks for sharing, grad students!), so when I plated the transformants smelled pretty awful. Hopefully that doesn’t make too much of
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              a difference.
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              <br/>
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          </p></p>
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      </div>
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    </div>
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    <div class="row day-entry">
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      <div class="columns small-2 small-offset-1 day-date">
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        <span>June</span>
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        <p>15</p>
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      </div>
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      <div class="columns small-9">
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        <h5>Innoculation of transformed colonies</h5>
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              <p dir="ltr">
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                  Recap from previous day: Colonies on all transformations were successful. Interestingly, the 1:50 dilution of DNA produced the largest number of
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                  colonies. All plates were very full, with the 1x plating dilutions being entirely unusable due to how covered they were. This may also be due to the
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                  long saving time from yesterday.
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              </p>
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              <br/>
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              <p dir="ltr">
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                  I decided to pick colonies from the 10x plating dilution of the 1:50 transformation.
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              </p>
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              <br/>
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              <p dir="ltr">
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                  <img
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                      src="https://lh4.googleusercontent.com/IbL6_FvBqLeLQZ2CUWZ2kXBGPqeqdqYRuUVUNAb4bbQ1elGA0cz5otVkYdDwt-nZ6nmpuJntbJSem5A7BDB00Cv71voqJRoKUOCsEPAf6to6G8YrJ7CX5U02RgD-vEmrwroCwDqy"
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                      width="234"
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−
                      height="312"
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−
                  />
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                  <img
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                      src="https://lh3.googleusercontent.com/rZ9_OctaSCPi6gFttR7WQl3rrlPa3ZWhmOvBJIu-mNOZNvh492n8usLOgfwTuPbRfXlx58zaeBzetvGkfcD23f9z-Twojg-8kjTF7iuAkkig2a37MsC_kC3Pqk_f_oNhHs2-GH6p"
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                      width="237"
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                      height="315"
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                  />
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              </p>
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              <br/>
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              <p dir="ltr">
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                  Goals: The only thing I’ll really be able to do is pick colonies and grow them overnight for tomorrow.
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              </p>
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              <br/>
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              <p dir="ltr">
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                  Inoculation:
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              </p>
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              <ul>
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                  <li dir="ltr">
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                      <p dir="ltr">
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                          Autoclaved culture tubes
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                      </p>
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                  </li>
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                  <li dir="ltr">
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                      <p dir="ltr">
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                          2 isolated colonies picked
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                      </p>
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                  </li>
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                  <li dir="ltr">
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                      <p dir="ltr">
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                          5mL LB per culture tube
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                      </p>
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                  </li>
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              </ul>
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              <br/>
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              <p dir="ltr">
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                  **Tubes in 37C incubator from 3:30pm
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              </p>
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              <br/>
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              Conclusions: The commercial cells were far more successful than our old cells. I used two colonies, which may not end up perfect, so I’ve stored the plate
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              in a 4C cooler. Tomorrow, I’ll store the cells and/or miniprep and possibly make glycerol stocks of them, though I don’t have primers to sequence or PCR
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              them beyond that.
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              <br/>
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      </div>
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    </div>
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<!-- PCAGE NEW DAY BEFORE THIS LINE --!>
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  </div>
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</div>
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<!-- PCAGE NEW WEEK BEFORE THIS LINE --!>
+
  
 
   </div>
 
   </div>

Revision as of 03:27, 7 October 2016

JOURNAL

P R O J E C T S

CDI

Protein Cages

Week

1

Construction of ATCC 13048 Loci 2 in pSB1C3

Isolation of template DNA from EC93, EC869, and ATCC 13048; PCR of Gibson fragments for EBL1 and EBL2 in pSB1C3 construct

Jun

27

Inoculation of strains obtained from Dr. Christopher Hayes

Inoculated overnight cultures of:

  • EPI100 w/ EC869 cosmid
  • X90 w/ EC93 cosmid
  • ATCC 13048

Conditions:

  • 10mL LB
  • 10uL ampicillin
  • 37 C for 16h shaking
Jun

28

Isolation of template DNA

Miniprep of EC93 and EC869

  • EC93 #1: 145.42 ng/μL
  • EC93 #2: 198.85 ng/μL
  • EC869 #1: 90.81 ng/μL
  • EC869 #2: 45.95 ng/μL

gDNA isolation of ATCC 13048

  • 34.87 ng/μL

Gibson primers designed for EBL1 and EBL2 toxin switched constructs in pSB1C3 and pSC101

Jun

29

PCR of Gibson fragments for EBL1 and EBL2 in pSB1C3

50uL rxn:

  • 2.5 uL F primer
  • 2.5 uL R primer
  • 25 uL Q5 2X MM
  • 19 uL dH2O

Fragments:

  • EBL1 in pSB1C3 frag 2
  • EBL2 in pSB1C3 frag 2
  • EBL2 in pSB1C3 frag 3

Cycling conditions:

  • 98C -- 30s
  • 98C -- 10s
  • TmC -- 30s
  • 72C -- ext time
    •
  • 72C -- 2min

28 cycles

Jun

30

Troubleshooting PCR of Gibson fragments
  • try using KAPA Hifi instead of Q5
  • add more template gDNA (1ng, 10ng, 35ng, 70ng)

    • 25uL rxn:

    • 0.75 uL F primer
    • 0.75 uL R primer
    • 12.5 uL KAPA Hifi 2X MM
    • dH2O up to 25 uL

    Fragments:

    • EBL1 in pSB1C3 frag 2 (Tm = 66C, ext time = 140s)
    • EBL2 in pSB1C3 frag 2 (Tm = 65C, ext time = 88s)
    • EBL2 in pSB1C3 frag 3 (Tm = 65C, ext time = 180s)

    Cycling conditions:

    • 98C -- 30s
    • 98C -- 10s
    • TmC -- 30s
    • 72C -- ext time
      •
    • 72C -- 2min

    28 cycles

    ladder; EBL2 frag 2; EBL1 frag 2; EBL2 frag 3

Week

2

Construction of ATCC 13048 Loci 2 in pSB1C3 Cont.

PCR of Gibson fragments for EBL1 and EBL2 in pSB1C3 construct

Jul

5

PCR of EBL1 and EBL2 Gibson fragments for pSB1C3

    Redoing failed PCR, modified protocol by having elongation time be extended to be 30s/kb; annealing temperature set to 72C; ran 25uL and 50uL rxn

    Fragments:

    • EBL1 in pSB1C3 frag 2 (Tm = 66C, ext time = 210s)
    • EBL2 in pSB1C3 frag 1 (Tm = 65C, ext time = 180s)
    • EBL2 in pSB1C3 frag 4 (Tm = 64C, ext time = 120s)

    Cycling conditions:

    • 98C -- 30s
    • 98C -- 10s
    • Tm -- 30s
    • 72C -- ext time
      •
    • 72C -- 2min
    • 14C -- infinite

    28 cycles

    ladder; EBL2 frag 1; EBL2 frag 4; EBL1 frag 2 (25uL); EBL1 frag 2 (50uL)

  • only primer dimers observed

Jul

6

Troubleshooting PCR of EBL1 frag 2, EBL2 frag 1, EBL2 frag 4

    Redoing failed PCR, modified protocol by doubling and quadrupling template dna amount; changed from 28 to 32 cycles

  • cycling conditions remained constant

    • ladder; 2x EBL1 frag 2; 4x EBL1 frag 2; 2x EBL1 frag 1; 4 EBL1 frag 1; 2x EBL1 frag 4; 4x EBL1 frag 4

  • doubling template DNA resolved EBL2 frag 2 and 4 problem; most likely faulty primers for EBL1 frag 2

Jul

7

Transformation of DH5-alpha with EC93 cosmid and pSK33
  • transform with 1ng of DNA
  • EC93: AmpR; pSK33: KanR
    • Jul

    8

    Troubleshooting PCR of EBL1 frag 2 in pSB1C3 Cont.
  • redid protocol in 7/6/16 with newly diluted primers

    • inoculated starter cultures of DH5-alpha w/ EC93, DH5-alpha w/ pSK33, and X90 w/ EC93

    Jul

    9

    Verification of EC93 cosmid and miniprepping for more pSK33
  • pSK33 had poor yield since it is a pSC101 derivative and is very low copy --> next time miniprep 10mL of culture

    • ran test digest to confirm EC93 cosmid:

    • 500 ng DNA
    • 5uL NEB 2.1
    • 1uL EcoRI
    • H2O to 50uL

    expecting bands at 10kb and 8kb

    ladder;EC93 cosmid from DH5-alpha;EC93 cosmid from X90;ladder