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| | <div class="row" id="cdi-team-journal"> | | <div class="row" id="cdi-team-journal"> |
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| − | <div class="week-entry"> | + | <iframe src="https://docs.google.com/document/d/166UOTOAKzmmcuEpqIt9wbnfjGKO3UwfefIOkTcaB-lI/pub?embedded=true" width="100%" style="height: 100vh;"></iframe> |
| − | <div class="row week-header" id="cdi-week-1-header">
| + | |
| − | <div class="columns small-2 week-number">
| + | |
| − | <span>Week</span>
| + | |
| − | <p>1</p>
| + | |
| − | </div>
| + | |
| − | <div class="columns small-10">
| + | |
| − | <h4> Construction of ATCC 13048 Loci 2 in pSB1C3</h4>
| + | |
| − | <p> Isolation of template DNA from EC93, EC869, and ATCC 13048; PCR of Gibson fragments for EBL1 and EBL2 in pSB1C3 construct </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="row week-content" id="cdi-week-1-content">
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| − | | + | |
| − | <div class="row day-entry">
| + | |
| − | <div class="columns small-2 small-offset-1 day-date">
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| − | <span>Jun</span>
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| − | <p>27</p>
| + | |
| − | </div>
| + | |
| − | <div class="columns small-9">
| + | |
| − | <h5>Inoculation of strains obtained from Dr. Christopher Hayes</h5>
| + | |
| − | <p> Inoculated overnight cultures of: </p>
| + | |
| − | <ul>
| + | |
| − | <li>EPI100 w/ EC869 cosmid</li>
| + | |
| − | <li>X90 w/ EC93 cosmid</li>
| + | |
| − | <li>ATCC 13048</li>
| + | |
| − | </ul>
| + | |
| − | | + | |
| − | <p> Conditions: </p>
| + | |
| − | <ul>
| + | |
| − | <li>10mL LB</li>
| + | |
| − | <li>10uL ampicillin</li>
| + | |
| − | <li>37 C for 16h shaking</li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="row day-entry">
| + | |
| − | <div class="columns small-2 small-offset-1 day-date">
| + | |
| − | <span>Jun</span>
| + | |
| − | <p>28</p>
| + | |
| − | </div>
| + | |
| − | <div class="columns small-9">
| + | |
| − | <h5>Isolation of template DNA</h5>
| + | |
| − | <p> Miniprep of EC93 and EC869 </p>
| + | |
| − | <ul>
| + | |
| − | <li>EC93 #1: 145.42 ng/μL</li>
| + | |
| − | <li>EC93 #2: 198.85 ng/μL</li>
| + | |
| − | <li>EC869 #1: 90.81 ng/μL</li>
| + | |
| − | <li>EC869 #2: 45.95 ng/μL</li>
| + | |
| − | </ul>
| + | |
| − | <p> gDNA isolation of ATCC 13048 </p>
| + | |
| − | <ul>
| + | |
| − | <li> 34.87 ng/μL </li>
| + | |
| − | </ul>
| + | |
| − | <p> Gibson primers designed for EBL1 and EBL2 toxin switched constructs in pSB1C3 and pSC101</p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="row day-entry">
| + | |
| − | <div class="columns small-2 small-offset-1 day-date">
| + | |
| − | <span>Jun</span>
| + | |
| − | <p>29</p>
| + | |
| − | </div>
| + | |
| − | <div class="columns small-9">
| + | |
| − | <h5>PCR of Gibson fragments for EBL1 and EBL2 in pSB1C3</h5>
| + | |
| − | <p> 50uL rxn: </p>
| + | |
| − | <ul>
| + | |
| − | <li>2.5 uL F primer</li>
| + | |
| − | <li>2.5 uL R primer</li>
| + | |
| − | <li>25 uL Q5 2X MM</li>
| + | |
| − | <li>19 uL dH2O</li>
| + | |
| − | </ul>
| + | |
| − | <p> Fragments: </p>
| + | |
| − | <ul>
| + | |
| − | <li>EBL1 in pSB1C3 frag 2</li>
| + | |
| − | <li>EBL2 in pSB1C3 frag 2</li>
| + | |
| − | <li>EBL2 in pSB1C3 frag 3</li>
| + | |
| − | </ul>
| + | |
| − | <p> Cycling conditions: </p>
| + | |
| − | <ul>
| + | |
| − | <li> 98C -- 30s</li>
| + | |
| − | </ul>
| + | |
| − | <ul> <b>
| + | |
| − | <li> 98C -- 10s</li>
| + | |
| − | <li> TmC -- 30s</li>
| + | |
| − | <li> 72C -- ext time</li>
| + | |
| − | </b> </ul>
| + | |
| − | <ul>
| + | |
| − | <li> 72C -- 2min</li>
| + | |
| − | </ul>
| + | |
| − | <p> 28 cycles </p>
| + | |
| − |
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="row day-entry">
| + | |
| − | <div class="columns small-2 small-offset-1 day-date">
| + | |
| − | <span>Jun</span>
| + | |
| − | <p>30</p>
| + | |
| − | </div>
| + | |
| − | <div class="columns small-9">
| + | |
| − | <h5>Troubleshooting PCR of Gibson fragments</h5>
| + | |
| − | <ul>
| + | |
| − | <li>try using KAPA Hifi instead of Q5</li>
| + | |
| − | <li>add more template gDNA (1ng, 10ng, 35ng, 70ng)</li>
| + | |
| − | <p> 25uL rxn: </p>
| + | |
| − | <ul>
| + | |
| − | <li>0.75 uL F primer</li>
| + | |
| − | <li>0.75 uL R primer</li>
| + | |
| − | <li>12.5 uL KAPA Hifi 2X MM</li>
| + | |
| − | <li>dH2O up to 25 uL</li>
| + | |
| − | </ul>
| + | |
| − | <p> Fragments: </p>
| + | |
| − | <ul>
| + | |
| − | <li>EBL1 in pSB1C3 frag 2 (Tm = 66C, ext time = 140s)</li>
| + | |
| − | <li>EBL2 in pSB1C3 frag 2 (Tm = 65C, ext time = 88s)</li>
| + | |
| − | <li>EBL2 in pSB1C3 frag 3 (Tm = 65C, ext time = 180s)</li>
| + | |
| − | </ul>
| + | |
| − | <p> Cycling conditions: </p>
| + | |
| − | <ul>
| + | |
| − | <li> 98C -- 30s</li>
| + | |
| − | </ul>
| + | |
| − | <ul> <b>
| + | |
| − | <li> 98C -- 10s</li>
| + | |
| − | <li> TmC -- 30s</li>
| + | |
| − | <li> 72C -- ext time</li>
| + | |
| − | </b> </ul>
| + | |
| − | <ul>
| + | |
| − | <li> 72C -- 2min</li>
| + | |
| − | </ul>
| + | |
| − | <p> 28 cycles </p>
| + | |
| − | <img src = "https://static.igem.org/mediawiki/2016/f/f6/Gel_7_1_16.png">
| + | |
| − | <p> ladder; EBL2 frag 2; EBL1 frag 2; EBL2 frag 3 </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <!-- CDI NEW DAY BEFORE THIS LINE --!>
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| − | | + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="week-entry">
| + | |
| − | <div class="row week-header" id="cdi-week-2-header">
| + | |
| − | <div class="columns small-2 week-number">
| + | |
| − | <span>Week</span>
| + | |
| − | <p>2</p>
| + | |
| − | </div>
| + | |
| − | <div class="columns small-10">
| + | |
| − | <h4> Construction of ATCC 13048 Loci 2 in pSB1C3 Cont.</h4>
| + | |
| − | <p> PCR of Gibson fragments for EBL1 and EBL2 in pSB1C3 construct </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="row week-content" id="cdi-week-2-content">
| + | |
| − | | + | |
| − | <div class="row day-entry">
| + | |
| − | <div class="columns small-2 small-offset-1 day-date">
| + | |
| − | <span>Jul</span>
| + | |
| − | <p>5</p>
| + | |
| − | </div>
| + | |
| − | <div class="columns small-9">
| + | |
| − | <h5>PCR of EBL1 and EBL2 Gibson fragments for pSB1C3</h5>
| + | |
| − | <ul>
| + | |
| − | <p>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</p>
| + | |
| − | | + | |
| − | <p> Fragments: </p>
| + | |
| − | <ul>
| + | |
| − | <li>EBL1 in pSB1C3 frag 2 (Tm = 66C, ext time = 210s)</li>
| + | |
| − | <li>EBL2 in pSB1C3 frag 1 (Tm = 65C, ext time = 180s)</li>
| + | |
| − | <li>EBL2 in pSB1C3 frag 4 (Tm = 64C, ext time = 120s)</li>
| + | |
| − | </ul>
| + | |
| − | <p> Cycling conditions: </p>
| + | |
| − | <ul> <li>98C -- 30s</li>
| + | |
| − | <b>
| + | |
| − | <li>98C -- 10s</li>
| + | |
| − | <li>Tm -- 30s</li>
| + | |
| − | <li>72C -- ext time</li>
| + | |
| − | </b>
| + | |
| − | <li>72C -- 2min</li>
| + | |
| − | <li>14C -- infinite</li> </ul>
| + | |
| − | <p> 28 cycles </p>
| + | |
| − | <img src = "https://static.igem.org/mediawiki/2016/9/95/EBL2_frag_1_4_EBL1_frag_2.png">
| + | |
| − | <p> ladder; EBL2 frag 1; EBL2 frag 4; EBL1 frag 2 (25uL); EBL1 frag 2 (50uL)</p>
| + | |
| − | <li> only primer dimers observed</p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="row day-entry">
| + | |
| − | <div class="columns small-2 small-offset-1 day-date">
| + | |
| − | <span>Jul</span>
| + | |
| − | <p>6</p>
| + | |
| − | </div>
| + | |
| − | <div class="columns small-9">
| + | |
| − | <h5>Troubleshooting PCR of EBL1 frag 2, EBL2 frag 1, EBL2 frag 4</h5>
| + | |
| − | <ul>
| + | |
| − | <p>Redoing failed PCR, modified protocol by doubling and quadrupling template dna amount; changed from 28 to 32 cycles</p>
| + | |
| − | <li> cycling conditions remained constant </li>
| + | |
| − | <img src = "https://static.igem.org/mediawiki/2016/5/5e/EBL1_frag_2_and_EBL2_frag_1%2C_4.png">
| + | |
| − | <p> 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</p>
| + | |
| − | <li> doubling template DNA resolved EBL2 frag 2 and 4 problem; most likely faulty primers for EBL1 frag 2 </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="row day-entry">
| + | |
| − | <div class="columns small-2 small-offset-1 day-date">
| + | |
| − | <span>Jul</span>
| + | |
| − | <p>7</p>
| + | |
| − | </div>
| + | |
| − | <div class="columns small-9">
| + | |
| − | <h5>Transformation of DH5-alpha with EC93 cosmid and pSK33</h5>
| + | |
| − | <li>transform with 1ng of DNA</li>
| + | |
| − | <li>EC93: AmpR; pSK33: KanR</li>
| + | |
| − |
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="row day-entry">
| + | |
| − | <div class="columns small-2 small-offset-1 day-date">
| + | |
| − | <span>Jul</span>
| + | |
| − | <p>8</p>
| + | |
| − | </div>
| + | |
| − | <div class="columns small-9">
| + | |
| − | <h5>Troubleshooting PCR of EBL1 frag 2 in pSB1C3 Cont. </h5>
| + | |
| − | <li>redid protocol in 7/6/16 with newly diluted primers</li>
| + | |
| − | <p> inoculated starter cultures of DH5-alpha w/ EC93, DH5-alpha w/ pSK33, and X90 w/ EC93 </p>
| + | |
| − |
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="row day-entry">
| + | |
| − | <div class="columns small-2 small-offset-1 day-date">
| + | |
| − | <span>Jul</span>
| + | |
| − | <p>9</p>
| + | |
| − | </div>
| + | |
| − | <div class="columns small-9">
| + | |
| − | <h5>Verification of EC93 cosmid and miniprepping for more pSK33</h5>
| + | |
| − | <li>pSK33 had poor yield since it is a pSC101 derivative and is very low copy --> next time miniprep 10mL of culture</li>
| + | |
| − | <p>ran test digest to confirm EC93 cosmid:</p>
| + | |
| − | <ul>
| + | |
| − | <li> 500 ng DNA </li>
| + | |
| − | <li> 5uL NEB 2.1 </li>
| + | |
| − | <li> 1uL EcoRI </li>
| + | |
| − | <li> H2O to 50uL </li>
| + | |
| − | </ul>
| + | |
| − | <p> expecting bands at 10kb and 8kb </p>
| + | |
| − | <img src ="https://static.igem.org/mediawiki/2016/1/19/Test_digest_EC93.jpeg">
| + | |
| − | <p> ladder;EC93 cosmid from DH5-alpha;EC93 cosmid from X90;ladder</p>
| + | |
| − |
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <!-- CDI NEW DAY BEFORE THIS LINE --!>
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| − | | + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <!-- CDI NEW WEEK BEFORE THIS LINE --!>
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| − | | + | |
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| | </div> | | </div> |
