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| | <div class="row team-select"> | | <div class="row team-select"> |
| − | <h2>Projects</h2> | + | <div class="team-select-header">P R O J E C T S</div> |
| | <div class="columns small-6 team team-active" id="team-cdi"> | | <div class="columns small-6 team team-active" id="team-cdi"> |
| | <h3>CDI</h3> | | <h3>CDI</h3> |
| | </div> | | </div> |
| − | <div class="columns small-6 team" id="team-pcage"> | + | <div class="columns small-6 team team-inactive" id="team-pcage"> |
| | <h3>Protein Cages</h3> | | <h3>Protein Cages</h3> |
| | </div> | | </div> |
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| | <div class="row" id="cdi-team-journal"> | | <div class="row" id="cdi-team-journal"> |
| | | | |
| − | <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 EBL2 in pSB1C3 construct </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="row week-content" id="cdi-week-1-content">
| + | |
| − | | + | |
| − | <div class="row day-entry">
| + | |
| − | <div class="columns small-2 small-offset-1 day-date">
| + | |
| − | <span>Jun</span>
| + | |
| − | <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/ EC93 cosmid</li>
| + | |
| − | <li>X90 w/ EC869 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 --!>
| + | |
| | | | |
| | </div> | | </div> |
| − | </div>
| |
| − |
| |
| − |
| |
| − | <!-- CDI NEW WEEK BEFORE THIS LINE --!>
| |
| − |
| |
| − |
| |
| − | </div>
| |
| − |
| |
| | | | |
| | <div class="row" id="pcage-team-journal"> | | <div class="row" id="pcage-team-journal"> |
| | | | |
| − | <div class="week-entry"> | + | <iframe src="https://docs.google.com/document/d/1ph7-1ltoQKndf_6v_vNtL0WnIjFDDo2TgkQmsf73iEg/pub?embedded=true" width="100%" style="height: 100vh;"></iframe> |
| − | <div class="row week-header" id="pcage-week-1-header">
| + | |
| − | <div class="columns small-2 week-number">
| + | |
| − | <span>Week</span>
| + | |
| − | <p>1</p>
| + | |
| − | </div>
| + | |
| − | <div class="columns small-10">
| + | |
| − | <h4>Title For The Week</h4>
| + | |
| − | <p>Description blah blah blah</p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="row week-content" id="pcage-week-1-content">
| + | |
| − | | + | |
| − | <div class="row day-entry">
| + | |
| − | <div class="columns small-2 small-offset-1 day-date">
| + | |
| − | <span>June</span>
| + | |
| − | <p>13</p>
| + | |
| − | </div>
| + | |
| − | <div class="columns small-9">
| + | |
| − | <h5>Plate Making and Transformation of O3-33 WT</h5>
| + | |
| − | <p><p dir="ltr">
| + | |
| − | Goals for today: We have a vector of O333 from Neil King contained in a pET vector, the goal for today is to transform this vector, grow it tomorrow and
| + | |
| − | PCR it for a large number of copies to work with and creation of a biobrick. Primers will need to be designed including the T7 promoter to terminator, with
| + | |
| − | iGEM prefix and suffix.
| + | |
| − | </p>
| + | |
| − | <br/>
| + | |
| − | <p dir="ltr">
| + | |
| − | Made plates
| + | |
| − | </p>
| + | |
| − | <ul>
| + | |
| − | <li dir="ltr">
| + | |
| − | <p dir="ltr">
| + | |
| − | 12.5g LB
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | <li dir="ltr">
| + | |
| − | <p dir="ltr">
| + | |
| − | 7.5 Bacto Agar
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | <li dir="ltr">
| + | |
| − | <p dir="ltr">
| + | |
| − | 500mL dH2O
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | <li dir="ltr">
| + | |
| − | <p dir="ltr">
| + | |
| − | 500uL Kanomycin (50mg/mL)
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <br/>
| + | |
| − | <ul>
| + | |
| − | <li dir="ltr">
| + | |
| − | <p dir="ltr">
| + | |
| − | 20 minutes autoclave
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | <li dir="ltr">
| + | |
| − | <p dir="ltr">
| + | |
| − | 4 hours total time to solidify
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <p dir="ltr">
| + | |
| − | => 32 plates total
| + | |
| − | </p>
| + | |
| − | <br/>
| + | |
| − | <p dir="ltr">
| + | |
| − | Transformation
| + | |
| − | </p>
| + | |
| − | <ul>
| + | |
| − | <li dir="ltr">
| + | |
| − | <p dir="ltr">
| + | |
| − | 30uL iGEM electrocompetent cells
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | <li dir="ltr">
| + | |
| − | <p dir="ltr">
| + | |
| − | 1uL O333 vector 1:100 dilution
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | <li dir="ltr">
| + | |
| − | <p dir="ltr">
| + | |
| − | 970uL SOC save
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <br/>
| + | |
| − | <ul>
| + | |
| − | <li dir="ltr">
| + | |
| − | <p dir="ltr">
| + | |
| − | 45 minutes incubation @ 800rpm/37C
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | <li dir="ltr">
| + | |
| − | <p dir="ltr">
| + | |
| − | 1 and 1:10 dilution plating
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <p dir="ltr">
| + | |
| − | => 3 transformations total
| + | |
| − | </p>
| + | |
| − | <p dir="ltr">
| + | |
| − | => arc time: 5.5s, 5.6s, 5.6s
| + | |
| − | </p>
| + | |
| − | <br/>
| + | |
| − | <p dir="ltr">
| + | |
| − | **Plates in 37C inoculation from 6:40pm
| + | |
| − | </p>
| + | |
| − | <br/>
| + | |
| − | <p dir="ltr">
| + | |
| − | Conclusions: Transformations had a consistent arc time. Tomorrow morning plates will be checked for colonies and grown.
| + | |
| − | </p></p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | | + | |
| − | <div class="row day-entry">
| + | |
| − | <div class="columns small-2 small-offset-1 day-date">
| + | |
| − | <span>June</span>
| + | |
| − | <p>14</p>
| + | |
| − | </div>
| + | |
| − | <div class="columns small-9">
| + | |
| − | <h5>Transformed O3-33 WT</h5>
| + | |
| − | <p dir="ltr">
| + | |
| − | Recap from previous day: No colonies were shown from any of the transformations. Competent cells may have been of low quality (relatively long arc times).
| + | |
| − | </p>
| + | |
| − | <p dir="ltr">
| + | |
| − | <strong>
| + | |
| − | <br/>
| + | |
| − | </strong>
| + | |
| − | </p>
| + | |
| − | <p dir="ltr">
| + | |
| − | Goals: Today, commercial electro-competent cells are going to be transformed, and different concentrations of DNA will be tested for transformation as
| + | |
| − | well.
| + | |
| − | </p>
| + | |
| − | <p dir="ltr">
| + | |
| − | <strong>
| + | |
| − | <br/>
| + | |
| − | </strong>
| + | |
| − | </p>
| + | |
| − | <p dir="ltr">
| + | |
| − | Transformation:
| + | |
| − | </p>
| + | |
| − | <ul>
| + | |
| − | <li dir="ltr">
| + | |
| − | <p dir="ltr">
| + | |
| − | 3x 25uL commercial electrocompetent cells
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | <li dir="ltr">
| + | |
| − | <p dir="ltr">
| + | |
| − | 1uL of 1:5, 1:50, 1:100 serial dilutions of plasmid from Neil King
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | <li dir="ltr">
| + | |
| − | <p dir="ltr">
| + | |
| − | 970uL SOC save
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <p dir="ltr">
| + | |
| − | <strong>
| + | |
| − | <br/>
| + | |
| − | </strong>
| + | |
| − | </p>
| + | |
| − | <ul>
| + | |
| − | <li dir="ltr">
| + | |
| − | <p dir="ltr">
| + | |
| − | 60 minutes save (800rpm @ 37C), then additional 50 minutes at 37C incubator (no shaking) as I was waiting for plating beads to be autoclaved and
| + | |
| − | cooled
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | <li dir="ltr">
| + | |
| − | <p dir="ltr">
| + | |
| − | 1 and 1:10 dilution platings
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <p dir="ltr">
| + | |
| − | => 1:5 DNA: 5.1s arc time
| + | |
| − | </p>
| + | |
| − | <p dir="ltr">
| + | |
| − | => 1:50 DNA: 5.0s arc time
| + | |
| − | </p>
| + | |
| − | <p dir="ltr">
| + | |
| − | => 1:100 DNA: 4.8s arc time
| + | |
| − | </p>
| + | |
| − | <p dir="ltr">
| + | |
| − | <strong>
| + | |
| − | <br/>
| + | |
| − | </strong>
| + | |
| − | </p>
| + | |
| − | <p dir="ltr">
| + | |
| − | **Plates in 37C incubation from 4:10pm
| + | |
| − | </p>
| + | |
| − | <p dir="ltr">
| + | |
| − | <br/>
| + | |
| − | Conclusions: Same as yesterday, though today arc times were slightly better. Unfortunately, my timing was off for plating because I was waiting to
| + | |
| − | autoclave new beads (thanks for sharing, grad students!), so when I plated the transformants smelled pretty awful. Hopefully that doesn’t make too much of
| + | |
| − | a difference.
| + | |
| − | <br/>
| + | |
| − | </p></p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | | + | |
| − | <div class="row day-entry">
| + | |
| − | <div class="columns small-2 small-offset-1 day-date">
| + | |
| − | <span>June</span>
| + | |
| − | <p>15</p>
| + | |
| − | </div>
| + | |
| − | <div class="columns small-9">
| + | |
| − | <h5>Innoculation of transformed colonies</h5>
| + | |
| − | <p><h1 dir="ltr">
| + | |
| − | <p dir="ltr">
| + | |
| − | Recap from previous day: Colonies on all transformations were successful. Interestingly, the 1:50 dilution of DNA produced the largest number of
| + | |
| − | colonies. All plates were very full, with the 1x plating dilutions being entirely unusable due to how covered they were. This may also be due to the
| + | |
| − | long saving time from yesterday.
| + | |
| − | </p>
| + | |
| − | <br/>
| + | |
| − | <p dir="ltr">
| + | |
| − | I decided to pick colonies from the 10x plating dilution of the 1:50 transformation.
| + | |
| − | </p>
| + | |
| − | <br/>
| + | |
| − | <p dir="ltr">
| + | |
| − | <img
| + | |
| − | src="https://lh4.googleusercontent.com/IbL6_FvBqLeLQZ2CUWZ2kXBGPqeqdqYRuUVUNAb4bbQ1elGA0cz5otVkYdDwt-nZ6nmpuJntbJSem5A7BDB00Cv71voqJRoKUOCsEPAf6to6G8YrJ7CX5U02RgD-vEmrwroCwDqy"
| + | |
| − | width="234"
| + | |
| − | height="312"
| + | |
| − | />
| + | |
| − | <img
| + | |
| − | src="https://lh3.googleusercontent.com/rZ9_OctaSCPi6gFttR7WQl3rrlPa3ZWhmOvBJIu-mNOZNvh492n8usLOgfwTuPbRfXlx58zaeBzetvGkfcD23f9z-Twojg-8kjTF7iuAkkig2a37MsC_kC3Pqk_f_oNhHs2-GH6p"
| + | |
| − | width="237"
| + | |
| − | height="315"
| + | |
| − | />
| + | |
| − | </p>
| + | |
| − | <br/>
| + | |
| − | <p dir="ltr">
| + | |
| − | Goals: The only thing I’ll really be able to do is pick colonies and grow them overnight for tomorrow.
| + | |
| − | </p>
| + | |
| − | <br/>
| + | |
| − | <p dir="ltr">
| + | |
| − | Inoculation:
| + | |
| − | </p>
| + | |
| − | <ul>
| + | |
| − | <li dir="ltr">
| + | |
| − | <p dir="ltr">
| + | |
| − | Autoclaved culture tubes
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | <li dir="ltr">
| + | |
| − | <p dir="ltr">
| + | |
| − | 2 isolated colonies picked
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | <li dir="ltr">
| + | |
| − | <p dir="ltr">
| + | |
| − | 5mL LB per culture tube
| + | |
| − | </p>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <br/>
| + | |
| − | <p dir="ltr">
| + | |
| − | **Tubes in 37C incubator from 3:30pm
| + | |
| − | </p>
| + | |
| − | <br/>
| + | |
| − | Conclusions: The commercial cells were far more successful than our old cells. I used two colonies, which may not end up perfect, so I’ve stored the plate
| + | |
| − | in a 4C cooler. Tomorrow, I’ll store the cells and/or miniprep and possibly make glycerol stocks of them, though I don’t have primers to sequence or PCR
| + | |
| − | them beyond that.
| + | |
| − | <br/>
| + | |
| − | </h1></p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | | + | |
| − | <!-- PCAGE NEW DAY BEFORE THIS LINE --!>
| + | |
| − | | + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <!-- PCAGE NEW WEEK BEFORE THIS LINE --!>
| + | |
| | | | |
| | </div> | | </div> |
