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| | <h1>Tuesday, August 30<sup>th</sup></h3> | | <h1>Tuesday, August 30<sup>th</sup></h3> |
| − | <h2>Results:</h2>
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| − | <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>
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| − | <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>
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| − | <div class="row">
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| − | <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=""/>
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| − | <p><strong>Figure 1:</strong> GFP transformation from iGEM kit</p>
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| − | </div>
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| − | <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=""/>
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| − | <p><strong>Figure 2:</strong> Gibson positive control on Amp</p>
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| − | </div>
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| − | </div>
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| − | <h2>Tasks:</h2>
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| − | <div class="row">
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| − | <div class="col-sm-2">
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| − | <p>Jordan</p>
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| − | </div>
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| − | <div class="col-sm-10">
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| − | <ul>
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| − | <li>Made Cam and Tet plates from the LB Kelly gave us</li>
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| − | <ul>
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| − | <li>Two bottles of 250 mL each</li>
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| − | <li>Added 3.75 grams of Bacto agar to each bottle and autoclaved, cooled to 55°C in water bath</li>
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| − | <li>Added 250 uL of Cam and Tet to respective media</li>
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| − | <li>Poured approximately 20 plates of each</li>
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| − | <li>Wrapped in foil and placed in the fridge </li>
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| − | </ul>
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| − | </ul>
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| − | </div>
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| − | </div>
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| − | <div class="row">
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| − | <div class="col-sm-2">
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| − | <p>Michelle</p>
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| − | </div>
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| − | <div class="col-sm-10">
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| − | <ul>
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| − | <li>Troubleshot transformation plating
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| − | <div class="row">
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| − | <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>
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| − | </div>
| + | |
| − | </li>
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| − | <li>Started running a western blot on TetR-Cas9 and Assembled Cas9 (in triplicate) with Ben from the Jewett lab</li>
| + | |
| − | </ul>
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| − | </div>
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| − | </div>
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| − | <div class="row">
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| − | <div class="col-sm-2">
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| − | <p>Sam</p>
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| − | </div>
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| − | <div class="col-sm-10">
| + | |
| − | <ul>
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| − | <li>Worked on questions for Dr. Postelnick</li>
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| − | <li>To do: set up a meeting (or maybe just email?) Dr. Tullman-Ercek</li>
| + | |
| − | <ul>
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| − | <li>Ask whether she knows of any good delivery mechanisms to get large proteins to the periplasm</li>
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| − | <li>Cas9 size</li>
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| − | <li>What pathways we’re already trying</li>
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| − | </ul>
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| − | </ul>
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| − | </div>
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| − | </div>
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| − | <div class="row">
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| − | <div class="col-sm-2">
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| − | <p>Tasfia</p>
| + | |
| − | </div>
| + | |
| − | <div class="col-sm-10">
| + | |
| − | <ul>
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| − | <li>Went to Jewett lab for western blotting TetR-Cas9 and Assembled Cas9</li>
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| − | <li>PCR: Linearization of tet backbone for GFP</li>
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| − | <ul>
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| − | <li>Three 50-uL reactions</li>
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| − | <li>1 uL OneTaq 2X Master Mix</li>
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| − | <li>1 uL DMSO</li>
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| − | <li>1 uL 10 uM forward primer</li>
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| − | <li>1 uL 10 uM reverse primer</li>
| + | |
| − | <li>1 uL template</li>
| + | |
| − | <ul>
| + | |
| − | <li>Template concentration: 28 ng/uL</li>
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| − | <li>Template amounts used:</li>
| + | |
| − | <ul>
| + | |
| − | <li>28 ng</li>
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| − | <li>2.8 ng</li>
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| − | <li>1.4 ng</li>
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| − | </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>
| + | |
| − | </li>
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| − | <li>Products are DpnI digesting at room temperature overnight</li>
| + | |
| − | </ul>
| + | |
| − | <li>PCR: Linearization of Cas9 for signal sequences</li>
| + | |
| − | <ul>
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| − | <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>
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| − | <li>25 uL OneTaq 2X Master Mix</li>
| + | |
| − | <li>1 uL DMSO </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>
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| − | <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>
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| − | <li>“Cas9 1:3”</li>
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| − | <ul>
| + | |
| − | <li>15 ng</li>
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| − | <li>1.5 ng</li>
| + | |
| − | </ul>
| + | |
| − | <li>“Cas9 miniprep w/ antibiotic”</li>
| + | |
| − | <ul>
| + | |
| − | <li>3.0 ng</li>
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| − | <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>
| + | |
| − | </div>
| + | |
| − | <div class="row">
| + | |
| − | <div class="col-sm-2">
| + | |
| − | <p>Tyler</p>
| + | |
| − | </div>
| + | |
| − | <div class="col-sm-10">
| + | |
| − | <ul>
| + | |
| − | <li>PCR: Linearization of mRFP</li>
| + | |
| − | <li>Aided in western blotting</li>
| + | |
| − | <li>Ordered Gel Kit & SybrSafe </li>
| + | |
| − | <li>Troubleshot nanodrop</li>
| + | |
| − | <li>Took some plate pics </li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </article>
| + | |
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