Difference between revisions of "Team:Northwestern/08 01"

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   <article>
 
   <article>
 
     <h1>Monday, August 1<sup>st</sup></h3>
 
     <h1>Monday, August 1<sup>st</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>Gibson reaction on 7.30 linearized tet backbone and Cas9 1+2
 +
            <ul>
 +
              <li>Insert:vector = 3:1</li>
 +
              <li>Backbone conc. 41 ng/uL</li>
 +
              <li>Used 50 ng backbone or 1.2 uL</li>
 +
              <li>Followed NEB kit protocol</li>
 +
              <li>Neg. Control- 1.2 ul vector + 8.8 ul water + 10 uL MasterMix</li>
 +
              <li>Positive control- 10 ul DNA provided in kit in 10 ul MasterMix</li>
 +
              <li>Incubated in water bath at 50 deg. 1 hour</li>
 +
            </ul>
 +
          </li>
 +
          <li>Transformed Gibson product
 +
            <ul>
 +
              <li>One test condition, four controls
 +
                <ul>
 +
                  <li>Actual Cas9 assembly—transformed 5 uL</li>
 +
                  <li>Negative control Gibson with backbone only, should get no or few colonies—5 uL</li>
 +
                  <li>Positive control from Gibson kit, should get colonies—5 uL</li>
 +
                  <li>No DNA transformed, should not get colonies—1 uL</li>
 +
                  <li>J04450 in pSB1C3- positive control, should get colonies—1uL</li>
 +
                </ul>
 +
              </li>
 +
              <li>Plated 100 uL of each transformation</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>Ran gel of Sara and Sam's 7.31 GFP and mCherry PCR
 +
            <ul>
 +
              <li>50 uL PCR reaction per piece + 10 uL of 6X Blue Loading Dye</li>
 +
              <li>25 uL loaded in each well</li>
 +
              <li>2 ladders run per gel, 2uL of 2kb ladder + 6uL 6X Blue Loading Dye</li>
 +
              <li>Realized that Sara and Sam did not put template in, so the primer dimers on the gels made sense</li>
 +
            </ul>
 +
          </li>
 +
          <div class="row">
 +
            <div class="col-sm-6"><img src="https://static.igem.org/mediawiki/2016/2/2c/T--Northwestern--08_01_1.jpg" width="952" height="1024" alt=""/>
 +
              <p>&nbsp;</p>
 +
              <img src="https://static.igem.org/mediawiki/2016/8/80/T--Northwestern--08_01_3.png" width="952" height="1024" alt=""/></div>
 +
            <div class="col-sm-6"><img src="https://static.igem.org/mediawiki/2016/a/a1/T--Northwestern--08_01_2.png" width="952" height="1024" alt=""/></div>
 +
          </div>
 +
          <li>Re-ran the PCR of the Tet Backbone Linearization for GFP/mCherry with Tyler
 +
            <ul>
 +
              <li>2, 50µL reactions
 +
                <ul>
 +
                  <li>20 μL water</li>
 +
                  <li>1 μL DMSO</li>
 +
                  <li>2 μL tet backbone</li>
 +
                  <li>1 μL 10 μM fwd primer</li>
 +
                  <li>1 μL 10 μM rev primer</li>
 +
                  <li>25 μL OneTaq master mix</li>
 +
                </ul>
 +
              </li>
 +
              <li>Negative Control (Water)
 +
                <ul>
 +
                  <li>22 μL water</li>
 +
                  <li>1 μL DMSO</li>
 +
                  <li>1 μL fwd primer</li>
 +
                  <li>1 μL rev primer</li>
 +
                  <li>25 μL OneTaq master mix </li>
 +
                </ul>
 +
              </li>
 +
              <li>Same conditions as previous PCR, but increased the start and end annealing temperatures by 1°C</li>
 +
            </ul>
 +
          </li>
 +
          <li>Took notes on Parallax scrolling, image transitioning in HTML and CSS—Started website</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Paul</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Ran a gel of GFP and mCherry PCR product with Sam
 +
            <ul>
 +
              <li>4 wells per GFP/mCherry, 25 uL per well</li>
 +
              <li>mCherry seems to have worked, GFP did not</li>
 +
            </ul>
 +
            <div class="row">
 +
              <div class="col-sm-6"><img src="https://static.igem.org/mediawiki/2016/7/70/T--Northwestern--08_01_4.png" width="2753" height="3361" alt=""/>
 +
                <p>&nbsp;</p>
 +
              <img src="https://static.igem.org/mediawiki/2016/c/c1/T--Northwestern--08_01_6.png" width="2729" height="3361" alt=""/>              </div>
 +
              <div class="col-sm-6"><img src="https://static.igem.org/mediawiki/2016/4/48/T--Northwestern--08_01_5.png" width="2729" height="3397" alt=""/></div>
 +
            </div>
 +
          </li>
 +
          <li>Looked over Cas9 Signaling Sequence primers: lots of primer dimers-probably easier/better to synthesize whole things</li>
 +
          <li>Investigate other SS</li>
 +
          <li>Feedback during group discussion</li>
 +
          <li>Got caught up from weekend work </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Sam</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Autoclaved TAE</li>
 +
          <li> PCR of GFP/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>95°C (2:00) | 95°C (0:07), 51°C (0:10), 72°C (0:43) | 72°C (5:00) </li>
 +
            </ul>
 +
          </li>
 +
          <li>DpnI digest: 1 uL of DpnI added to each of the 50uL tubes. Incubated in the 37 for 4 hours</li>
 +
          <li>Ran gel on GFP/mCherry PCR with Paul</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Sara</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Ran a PCR of GFP and mCherry to put on the GG ends with Sam</li>
 +
          <li>Talked to Patrick to get more Dpn1 to use on the GFP mCherry PCR</li>
 +
          <li>Learned that we’ve been using too much Dpn1, and Patrick suggested using 1 uL per 100 uL tube</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Tasfia </p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
         
 +
          <li> PCR Tet Backbone Linearization for GFP/mCherry with Tyler
 +
            <ul>
 +
            <li>Reactions
 +
              <ul><li>10 μL water</li>
 +
              <li>0.5 μL DMSO</li>
 +
              <li>1 μL tet backbone</li>
 +
              <li>0.5 μL 10 μM fwd primer</li>
 +
              <li>0.5 μL 10 μM rev primer</li>
 +
              <li>12.5 μL OneTaq master mix</li>
 +
            </ul>
 +
          </li>
 +
         
 +
          <li>Negative control for 25-μL reaction (1 tube)
 +
          <ul><li>11 μL water</li>
 +
          <li>0.5 μL DMSO</li>
 +
          <li>0.5 μL fwd primer</li>
 +
          <li>0.5 μL rev primer</li>
 +
          <li>12.5 μL OneTaq master mix</li></ul>
 +
          <div class="row">
 +
              <div class="col-sm-6"><img src="https://static.igem.org/mediawiki/2016/5/55/T--Northwestern--08_01_7.png" width="711" height="173" alt=""/></div>
 +
            </div>
 +
            </li>
 +
            </ul></li>
 +
            <li>DpnI digest on PCR product (added 1 μL DpnI in each reaction tube)</li>
 +
            <li>Ran a gel on the PCR product of Tet Backbone Linearization for GFP/mCherry
 +
            <ul><li>Two 8-μL ladders on each side of gel; 1:3 purple 2-log ladder to 6X blue loading dye</li>
 +
            <li>Each well had ~28 μL PCR product + loading dye (~26 PCR product + ~5.2 μL 6X blue loading dye)</li>
 +
            <li>95 V</li>
 +
            <li>Ran for ~1 hour</li></ul>
 +
            <div class="row">
 +
              <div class="col-sm-6"><img src="https://static.igem.org/mediawiki/2016/e/e1/T--Northwestern--08_01_8.png"  alt=""/></div>
 +
            </div>
  
−
<footer id="nav">
+
            </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Tyler</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li> PCR Tet Backbone Linearization for GFP/mCherry with Thush</li>
 +
          <li>Gel on Tet linearization with Thush</li>
 +
          <li>PCR of the Tet Backbone Linearization for GFP/mCherry with Michelle</li>
 +
          <li>Reviewed SS cas9 parts</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
  </article><footer id="nav">
 
       <div class="row">
 
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         <div class="col-sm-4"><a href="https://2016.igem.org/Team:Northwestern/07_31"><img src="https://static.igem.org/mediawiki/2016/c/c6/T--Northwestern--backarrow.png" height="15" width="15"/> yesterday</a></div>
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Revision as of 16:11, 15 October 2016

Notebook

Monday, August 1st

Tasks:

Jordan

  • Gibson reaction on 7.30 linearized tet backbone and Cas9 1+2
    • Insert:vector = 3:1
    • Backbone conc. 41 ng/uL
    • Used 50 ng backbone or 1.2 uL
    • Followed NEB kit protocol
    • Neg. Control- 1.2 ul vector + 8.8 ul water + 10 uL MasterMix
    • Positive control- 10 ul DNA provided in kit in 10 ul MasterMix
    • Incubated in water bath at 50 deg. 1 hour
  • Transformed Gibson product
    • One test condition, four controls
      • Actual Cas9 assembly—transformed 5 uL
      • Negative control Gibson with backbone only, should get no or few colonies—5 uL
      • Positive control from Gibson kit, should get colonies—5 uL
      • No DNA transformed, should not get colonies—1 uL
      • J04450 in pSB1C3- positive control, should get colonies—1uL
    • Plated 100 uL of each transformation

Michelle

  • Ran gel of Sara and Sam's 7.31 GFP and mCherry PCR
    • 50 uL PCR reaction per piece + 10 uL of 6X Blue Loading Dye
    • 25 uL loaded in each well
    • 2 ladders run per gel, 2uL of 2kb ladder + 6uL 6X Blue Loading Dye
    • Realized that Sara and Sam did not put template in, so the primer dimers on the gels made sense

    •  

  • Re-ran the PCR of the Tet Backbone Linearization for GFP/mCherry with Tyler
    • 2, 50µL reactions
      • 20 μL water
      • 1 μL DMSO
      • 2 μL tet backbone
      • 1 μL 10 μM fwd primer
      • 1 μL 10 μM rev primer
      • 25 μL OneTaq master mix
    • Negative Control (Water)
      • 22 μL water
      • 1 μL DMSO
      • 1 μL fwd primer
      • 1 μL rev primer
      • 25 μL OneTaq master mix
    • Same conditions as previous PCR, but increased the start and end annealing temperatures by 1°C
  • Took notes on Parallax scrolling, image transitioning in HTML and CSS—Started website

Paul

  • Ran a gel of GFP and mCherry PCR product with Sam
    • 4 wells per GFP/mCherry, 25 uL per well
    • mCherry seems to have worked, GFP did not

     

  • Looked over Cas9 Signaling Sequence primers: lots of primer dimers-probably easier/better to synthesize whole things
  • Investigate other SS
  • Feedback during group discussion
  • Got caught up from weekend work

Sam

  • Autoclaved TAE
  • PCR of GFP/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
    • 95°C (2:00) | 95°C (0:07), 51°C (0:10), 72°C (0:43) | 72°C (5:00)
  • DpnI digest: 1 uL of DpnI added to each of the 50uL tubes. Incubated in the 37 for 4 hours
  • Ran gel on GFP/mCherry PCR with Paul

Sara

  • Ran a PCR of GFP and mCherry to put on the GG ends with Sam
  • Talked to Patrick to get more Dpn1 to use on the GFP mCherry PCR
  • Learned that we’ve been using too much Dpn1, and Patrick suggested using 1 uL per 100 uL tube

Tasfia

  • PCR Tet Backbone Linearization for GFP/mCherry with Tyler
    • Reactions
      • 10 μL water
      • 0.5 μL DMSO
      • 1 μL tet backbone
      • 0.5 μL 10 μM fwd primer
      • 0.5 μL 10 μM rev primer
      • 12.5 μL OneTaq master mix
    • Negative control for 25-μL reaction (1 tube)
      • 11 μL water
      • 0.5 μL DMSO
      • 0.5 μL fwd primer
      • 0.5 μL rev primer
      • 12.5 μL OneTaq master mix
  • DpnI digest on PCR product (added 1 μL DpnI in each reaction tube)
  • Ran a gel on the PCR product of Tet Backbone Linearization for GFP/mCherry
    • Two 8-μL ladders on each side of gel; 1:3 purple 2-log ladder to 6X blue loading dye
    • Each well had ~28 μL PCR product + loading dye (~26 PCR product + ~5.2 μL 6X blue loading dye)
    • 95 V
    • Ran for ~1 hour

Tyler

  • PCR Tet Backbone Linearization for GFP/mCherry with Thush
  • Gel on Tet linearization with Thush
  • PCR of the Tet Backbone Linearization for GFP/mCherry with Michelle
  • Reviewed SS cas9 parts