Difference between revisions of "Team:Northwestern/07 31"

Line 20: Line 20:
 
   <article>
 
   <article>
 
     <h1>Sunday, July 31<sup>st</sup></h3>
 
     <h1>Sunday, July 31<sup>st</sup></h3>
−
    <h2>Agenda:</h2>
+
<h2>Tasks:</h2>
−
 
+
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Sara &amp; Sam</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Reran (7/29) PCR Linearization of the Tet Backbone for Cas9 Gibson +DMSO
 +
            <ul>
 +
              <li> 4 tubes of the following recipe in order to increase concentration resulting from gel extract:
 +
                <ul>
 +
                  <li>10 uL water</li>
 +
                  <li>0.5 uL DMSO (used to increase purity of results)</li>
 +
                  <li>1.0 uL Tet template (from miniprep)</li>
 +
                  <li>0.5 uL 10uM fwd primer</li>
 +
                  <li>0.5 uL 10uM rev primer</li>
 +
                  <li>12.5 uL OneTaq Master Mix</li>
 +
                  <li>Total: 25 uL </li>
 +
                  <li>95&#176;C (2:00) | 95&#176;C (0:07), &nbsp;60&#176;C (0:10), &nbsp;72&#176;C (1:06) [10 cycles] | 95&#176;C (0:07), 50&#176;C (0:10), 72&#176;C (1:06) [18 cycles] | 72&#176;C (5:00), 4&#176;C (20:00)</li>
 +
                </ul>
 +
              </li>
 +
              <li>DpnI digest: 1 uL of DpnI added to each of the 4 tubes. Incubated in the 37 for one hour</li>
 +
              <li>Ran gel
 +
                <ul>
 +
                  <li>Loaded 20 uL of each of the 4 tubes of product into each well (we didn’t want to overload), and the other 5 uL into the next 4 </li>
 +
                  <li>All 4 are the same product and look to be about the right size, accounting for the wonkiness of the gel </li>
 +
                </ul>
 +
              </li>
 +
              <li>Gel extracted of the Linearized Tet Backbone- Gibson
 +
                <ul>
 +
                  <li>Concentration: 41.6 ng/uL </li>
 +
                </ul>
 +
              </li>
 +
            </ul>
 +
            <div class="row">
 +
              <div class="col-sm-6"><img src="https://static.igem.org/mediawiki/2016/c/c1/T--Northwestern--07_31_1.jpg" width="1385" height="1721" alt=""/></div>
 +
            </div>
 +
          </li>
 +
          <li>Reran PCR of GFP and mCherry
 +
            <ul>
 +
              <li>25 uL OneTaq</li>
 +
              <li>1 uL diluted 10 mM f primer</li>
 +
              <li>1 uL diluted 10 mM r primer</li>
 +
              <li>1 uL GFP/mCherry</li>
 +
              <li>21 uL dH20</li>
 +
              <li>1 uL DMSO</li>
 +
              <li>Ran 2 tubes for each GFP and mCherry at 50 uL volume, per Patrick’s advice</li>
 +
              <li>95&#176;C (2:00) | 95&#176;C (0:07), 51&#176;C (0:10), 72&#176;C (0:43) | 72&#176;C (5:00) </li>
 +
            </ul>
 +
          </li>
 +
          <li>DpnI digest
 +
            <ul>
 +
              <li>Incubated at 37&#176;C for 2 hours, then overnight</li>
 +
              <li>1 uL per 50 uL PCR reaction</li>
 +
            </ul>
 +
          </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
  </article>
 
<footer id="nav">
 
<footer id="nav">
 
       <div class="row">
 
       <div class="row">

Revision as of 15:24, 15 October 2016

Notebook

Sunday, July 31st

Tasks:

Sara & Sam

  • Reran (7/29) PCR Linearization of the Tet Backbone for Cas9 Gibson +DMSO
    • 4 tubes of the following recipe in order to increase concentration resulting from gel extract:
      • 10 uL water
      • 0.5 uL DMSO (used to increase purity of results)
      • 1.0 uL Tet template (from miniprep)
      • 0.5 uL 10uM fwd primer
      • 0.5 uL 10uM rev primer
      • 12.5 uL OneTaq Master Mix
      • Total: 25 uL
      • 95°C (2:00) | 95°C (0:07),  60°C (0:10),  72°C (1:06) [10 cycles] | 95°C (0:07), 50°C (0:10), 72°C (1:06) [18 cycles] | 72°C (5:00), 4°C (20:00)
    • DpnI digest: 1 uL of DpnI added to each of the 4 tubes. Incubated in the 37 for one hour
    • Ran gel
      • Loaded 20 uL of each of the 4 tubes of product into each well (we didn’t want to overload), and the other 5 uL into the next 4
      • All 4 are the same product and look to be about the right size, accounting for the wonkiness of the gel
    • Gel extracted of the Linearized Tet Backbone- Gibson
      • Concentration: 41.6 ng/uL
  • Reran PCR of GFP and mCherry
    • 25 uL OneTaq
    • 1 uL diluted 10 mM f primer
    • 1 uL diluted 10 mM r primer
    • 1 uL GFP/mCherry
    • 21 uL dH20
    • 1 uL DMSO
    • Ran 2 tubes for each GFP and mCherry at 50 uL volume, per Patrick’s advice
    • 95°C (2:00) | 95°C (0:07), 51°C (0:10), 72°C (0:43) | 72°C (5:00)
  • DpnI digest
    • Incubated at 37°C for 2 hours, then overnight
    • 1 uL per 50 uL PCR reaction