Difference between revisions of "Team:Northwestern/08 30"

 
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   <article>
 
   <article>
 
     <h1>Tuesday, August 30<sup>th</sup></h3>
 
     <h1>Tuesday, August 30<sup>th</sup></h3>
−
<h2>Results:</h2>
+
<h2>Tasks:</h2>
−
    <p> While GFP and the Gibson kit positive control had colonies, the gRNA Gibson product (Tet) and positive transformation control (Cam) did not yield colonies.</p>
+
−
    <p>The gRNA Gibson has falied grow the past two times we transformed it, so we’re suspicious that it might not be a problem with the Gibson assembly, because the Gibson control has grown on Amp.</p>
+
−
    <div class="row">
+
−
      <div class="col-xs-6"><img src="https://static.igem.org/mediawiki/2016/0/07/T--Northwestern--08_29_3.jpg" width="1200" height="1600" alt=""/>
+
−
        <p><strong>Figure 1:</strong> GFP transformation from iGEM kit</p>
+
−
      </div>
+
−
      <div class="col-xs-6"><img src="https://static.igem.org/mediawiki/2016/4/4b/T--Northwestern--08_29_5.jpg" width="1200" height="1600" alt=""/>
+
−
        <p><strong>Figure 2:</strong> Gibson positive control on Amp</p>
+
−
      </div>
+
−
    </div>
+
−
    <h2>Tasks:</h2>
+
 
     <div class="row">
 
     <div class="row">
 
       <div class="col-sm-2">
 
       <div class="col-sm-2">
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       <div class="col-sm-10">
 
       <div class="col-sm-10">
 
         <ul>
 
         <ul>
−
           <li>Made Cam and Tet plates from the LB Kelly gave us</li>
+
           <li>Started cytosolic GFP cultures for tomorrow</li>
−
           <ul>
+
           <li>Interviewed Dr. Postelnick</li>
−
            <li>Two bottles of 250 mL each</li>
+
          <li>Worked on travel grant</li>
−
            <li>Added 3.75 grams of Bacto agar to each bottle and autoclaved, cooled to 55&#176;C in water bath</li>
+
          <li>Read instructions for millipore filters </li>
−
            <li>Added 250 uL of Cam and Tet to respective media</li>
+
−
            <li>Poured approximately 20 plates of each</li>
+
−
            <li>Wrapped in foil and placed in the fridge </li>
+
−
          </ul>
+
 
         </ul>
 
         </ul>
 
       </div>
 
       </div>
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       <div class="col-sm-10">
 
       <div class="col-sm-10">
 
         <ul>
 
         <ul>
−
           <li>Troubleshot transformation plating
+
           <li>Ran western blot</li>
−
             <div class="row">
+
          <ul>
−
               <div class="col-xs-12"><img src="https://static.igem.org/mediawiki/2016/e/e5/T--Northwestern--08_29_4.png" width="1204" height="366" alt=""/></div>
+
             <li>We successfully expressed Cas9
−
            </div>
+
              <div class="row">
−
          </li>
+
                <div class="col-xs-8 col-sm-12"><img src="https://static.igem.org/mediawiki/2016/b/bd/T--Northwestern--08_30_1.png" width="1498" height="838" alt=""/></div>
−
          <li>Started running a western blot on TetR-Cas9 and Assembled Cas9 (in triplicate) with Ben from the Jewett lab</li>
+
              </div>
 +
               <div class="row">
 +
                <div class="col-xs-8 col-sm-12"><img src="https://static.igem.org/mediawiki/2016/6/67/T--Northwestern--08_30_2.png" width="1228" height="687" alt=""/></div>
 +
              </div>
 +
            </li>
 +
            <li>There are no bands in lanes 2-4 because Thush didn’t induce the promoter but Ben offered to run another Western after inducing</li>
 +
            <li>It was a good control</li>
 +
            <li>Cas9 seems to appear between the 97 and 191 kDa bands, but closer to the 97 kDa band, which is expected (~114 kDa is what we’re going for)</li>
 +
            <li>Each of the “Cas9 Assembled” lanes have a lot more bands than expected, and this could be attributed to truncated protein expression and cleavage sites</li>
 +
            <li>Kelly can give us cells we can run a western on as a control, to see if the extra bands on lanes 5-7 are attributed to noise </li>
 +
          </ul>
 
         </ul>
 
         </ul>
 
       </div>
 
       </div>
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       <div class="col-sm-10">
 
       <div class="col-sm-10">
 
         <ul>
 
         <ul>
−
           <li>Worked on questions for Dr. Postelnick</li>
+
           <li>Interviewed Dr. Postelnick</li>
−
           <li>To do: set up a meeting (or maybe just email?) Dr. Tullman-Ercek</li>
+
           <li>Emailed Dr. Tullman-Ercek</li>
−
           <ul>
+
           <li>Cited antibiotics pamphlet for adults</li>
−
            <li>Ask whether she knows of any good delivery mechanisms to get large proteins to the periplasm</li>
+
−
            <li>Cas9 size</li>
+
−
            <li>What pathways we’re already trying</li>
+
−
          </ul>
+
 
         </ul>
 
         </ul>
 
       </div>
 
       </div>
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       <div class="col-sm-10">
 
       <div class="col-sm-10">
 
         <ul>
 
         <ul>
−
           <li>Went to Jewett lab for western blotting TetR-Cas9 and Assembled Cas9</li>
+
           <li>DpnI digest of Cas9-Lrz-SS PCR products from 8.29.16</li>
−
           <li>PCR: Linearization of tet backbone for GFP</li>
+
           <li>Golden Gate PCR for sfGFP and mCherry (nine reactions, one 50-uL reaction for each) </li>
 
           <ul>
 
           <ul>
−
             <li>Three 50-uL reactions</li>
+
             <li>sfGFP taken from 8.23.16 miniprep</li>
−
             <li>1 uL OneTaq 2X Master Mix</li>
+
             <li>mCherry taken from 7.35.16 miniprep </li>
−
             <li>1 uL DMSO</li>
+
             <li>Amount of template used:
−
            <li>1 uL 10 uM forward primer</li>
+
               <div class="row">
−
            <li>1 uL 10 uM reverse primer</li>
+
                 <div class="col-xs-8 col-sm-6"><img src="https://static.igem.org/mediawiki/2016/4/49/T--Northwestern--08_30_3.png" width="1228" height="687" alt=""/></div>
−
            <li>1 uL template</li>
+
−
            <ul>
+
−
              <li>Template concentration: 28 ng/uL</li>
+
−
              <li>Template amounts used:</li>
+
−
               <ul>
+
−
                <li>28 ng</li>
+
−
                <li>2.8 ng</li>
+
−
                <li>1.4 ng</li>
+
−
              </ul>
+
−
            </ul>
+
−
            <li>Conditions:
+
−
            <div class="row">
+
−
                 <div class="col-xs-8 col-sm-12"><img src="https://static.igem.org/mediawiki/2016/2/26/T--Northwestern--08_29_2.png" width="1340" height="186" alt=""/></div>
+
 
               </div>
 
               </div>
 
             </li>
 
             </li>
−
             <li>Products are DpnI digesting at room temperature overnight</li>
+
             <li>Conditions: 95&#176;C (5:00) | 95&#176;C (0:07), 54&#176;C (0:10), 72&#176;C (0:43) | 72&#176;C (2:00)</li>
 
           </ul>
 
           </ul>
−
           <li>PCR: Linearization of Cas9 for signal sequences</li>
+
           <li>Ran gels on Tet-Lrz-GFP (8.29.16), Cas9-Lrz-SS (8.29.16), and all the GFP/mCherry for GG PCR products (8.30.16)</li>
 
           <ul>
 
           <ul>
−
             <li>We had to rerun the Cas9-Lrz-SS procedure because the gel extractions from the most recent PCR gave us erroneous concentrations and 260 ratios (~555 ng/uL, really high 260/280 and 260/230 ratios) </li>
+
             <li>Used SybrSafe from Jewett lab (bands were very faint on all gels)</li>
−
             <li>25 uL OneTaq 2X Master Mix</li>
+
             <li>Recommended: Use ~3 uL SybrSafe on small 10-well gels (with SybrGreen you can get away with ~1 uL)</li>
−
            <li>1 uL DMSO </li>
+
            <li>These gels used ~3 uL on the 14-well gels </li>
−
            <li>1 uL 10 μM forward primer</li>
+
−
            <li>1 uL 10 uM reverse primer</li>
+
−
            <li>1 uL template</li>
+
−
            <li>21 uL nuclease-free water </li>
+
−
            <li>Two Cas9 template samples used:</li>
+
−
            <ul>
+
−
              <li>“Cas9 1:3” (concentration ~155 ng/uL)</li>
+
−
              <li>“Cas9 miniprep w/ antibiotic” (concentration ~30 ng/uL)</li>
+
−
            </ul>
+
−
            <li>Four 50-uL reactions with the following template amounts</li>
+
−
            <ul>
+
−
              <li>“Cas9 1:3”</li>
+
−
              <ul>
+
−
                <li>15 ng</li>
+
−
                <li>1.5 ng</li>
+
−
              </ul>
+
−
              <li>“Cas9 miniprep w/ antibiotic”</li>
+
−
              <ul>
+
−
                <li>3.0 ng</li>
+
−
                <li>1.5 ng </li>
+
−
              </ul>
+
−
            </ul>
+
−
            <li>Conditions:
+
−
              <div class="row">
+
−
                <div class="col-xs-8 col-sm-12"><img src="https://static.igem.org/mediawiki/2016/e/e0/T--Northwestern--08_29_1.png" width="1340" height="186" alt=""/></div>
+
−
              </div>
+
−
            </li>
+
−
            <li>Product is in the thermal cycler and will be ready to DpnI digest tomorrow morning</li>
+
 
           </ul>
 
           </ul>
 +
          <div class="row">
 +
            <div class="col-xs-8 col-sm-6"><img src="https://static.igem.org/mediawiki/2016/9/9a/T--Northwestern--08_30_5.png" width="1228" height="687" alt=""/>
 +
              <p>2kB band is very faint</p>
 +
            </div>
 +
            <div class="col-xs-8 col-sm-6"><img src="https://static.igem.org/mediawiki/2016/1/11/T--Northwestern--08_30_6.png" width="1228" height="687" alt=""/></div>
 +
          </div>
 
         </ul>
 
         </ul>
 
       </div>
 
       </div>
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       <div class="col-sm-10">
 
       <div class="col-sm-10">
 
         <ul>
 
         <ul>
−
           <li>PCR: Linearization of mRFP</li>
+
           <li>Redesigned primers for restriction digestion of Cas9 into PSB1C3</li>
−
           <li>Aided in western blotting</li>
+
           <li>Looked into reusing primers for sequencing of gRNA</li>
−
           <li>Ordered Gel Kit &amp; SybrSafe </li>
+
           <li>Transformation test with Cam plates</li>
−
           <li>Troubleshot nanodrop</li>
+
           <ul>
−
           <li>Took some plate pics </li>
+
            <li>Cam and Tet plate test results</li>
 +
            <ul>
 +
              <li>Tet plates prepared with Kelly’s LB grew exponentially more colonies than tet plates prepared with our LB on August 8th</li>
 +
              <li>gRNA Gibson product grew one colony</li>
 +
              <li>Nothing grew on plates prepared with our LB, plates prepared with Kelly’s LB, or Kelly’s already-prepared plates from Leonard lab</li>
 +
              <li>There’s probably something wrong with our positive control plasmid for Cam</li>
 +
            </ul>
 +
          </ul>
 +
           <li>Drafted e-mail to Tullman-Ercek w/Sam </li>
 
         </ul>
 
         </ul>
 
       </div>
 
       </div>
 
     </div>
 
     </div>
 
   </article>
 
   </article>
 +
 
 
<footer id="nav">
 
<footer id="nav">
 
       <div class="row">
 
       <div class="row">
 
         <div class="col-sm-4"><a href="https://2016.igem.org/Team:Northwestern/08_29"><img src="https://static.igem.org/mediawiki/2016/c/c6/T--Northwestern--backarrow.png" height="15" width="15"/> yesterday</a></div>
 
         <div class="col-sm-4"><a href="https://2016.igem.org/Team:Northwestern/08_29"><img src="https://static.igem.org/mediawiki/2016/c/c6/T--Northwestern--backarrow.png" height="15" width="15"/> yesterday</a></div>
−
         <div class="col-sm-4"><a href="https://2016.igem.org/Team:Northwestern/Notebook">back to calender </a></div>
+
         <div class="col-sm-4"><a href="https://2016.igem.org/Team:Northwestern/Notebook">back to calendar </a></div>
 
         <div class="col-sm-4"><a href="https://2016.igem.org/Team:Northwestern/08_31">tomorrow <img src="https://static.igem.org/mediawiki/2016/6/65/T--Northwestern--forwardarrow.png" height="15" width="15"/></a></div>
 
         <div class="col-sm-4"><a href="https://2016.igem.org/Team:Northwestern/08_31">tomorrow <img src="https://static.igem.org/mediawiki/2016/6/65/T--Northwestern--forwardarrow.png" height="15" width="15"/></a></div>
 
       </div>
 
       </div>

Latest revision as of 15:34, 18 October 2016

Notebook

Tuesday, August 30th

Tasks:

Jordan

  • Started cytosolic GFP cultures for tomorrow
  • Interviewed Dr. Postelnick
  • Worked on travel grant
  • Read instructions for millipore filters

Michelle

  • Ran western blot
    • We successfully expressed Cas9
    • There are no bands in lanes 2-4 because Thush didn’t induce the promoter but Ben offered to run another Western after inducing
    • It was a good control
    • Cas9 seems to appear between the 97 and 191 kDa bands, but closer to the 97 kDa band, which is expected (~114 kDa is what we’re going for)
    • Each of the “Cas9 Assembled” lanes have a lot more bands than expected, and this could be attributed to truncated protein expression and cleavage sites
    • Kelly can give us cells we can run a western on as a control, to see if the extra bands on lanes 5-7 are attributed to noise

Sam

  • Interviewed Dr. Postelnick
  • Emailed Dr. Tullman-Ercek
  • Cited antibiotics pamphlet for adults

Tasfia

  • DpnI digest of Cas9-Lrz-SS PCR products from 8.29.16
  • Golden Gate PCR for sfGFP and mCherry (nine reactions, one 50-uL reaction for each)
    • sfGFP taken from 8.23.16 miniprep
    • mCherry taken from 7.35.16 miniprep
    • Amount of template used:
    • Conditions: 95°C (5:00) | 95°C (0:07), 54°C (0:10), 72°C (0:43) | 72°C (2:00)
  • Ran gels on Tet-Lrz-GFP (8.29.16), Cas9-Lrz-SS (8.29.16), and all the GFP/mCherry for GG PCR products (8.30.16)
    • Used SybrSafe from Jewett lab (bands were very faint on all gels)
    • Recommended: Use ~3 uL SybrSafe on small 10-well gels (with SybrGreen you can get away with ~1 uL)
    • These gels used ~3 uL on the 14-well gels

    2kB band is very faint

Tyler

  • Redesigned primers for restriction digestion of Cas9 into PSB1C3
  • Looked into reusing primers for sequencing of gRNA
  • Transformation test with Cam plates
    • Cam and Tet plate test results
      • Tet plates prepared with Kelly’s LB grew exponentially more colonies than tet plates prepared with our LB on August 8th
      • gRNA Gibson product grew one colony
      • Nothing grew on plates prepared with our LB, plates prepared with Kelly’s LB, or Kelly’s already-prepared plates from Leonard lab
      • There’s probably something wrong with our positive control plasmid for Cam
  • Drafted e-mail to Tullman-Ercek w/Sam