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| − | <img src="https://static.igem.org/mediawiki/2016/e/e4/T--Imperial_College--ProtoBanner.png" height="450"/>
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| − | <p>On this page, the ecolibrium team would like to share with you the protocols that we have been using over the summer. This library of protocols has been developed alongside our supervisors for the purpose of this study. Here you can find the exact methods we use to generate our data and results. We share them in the interest of reproducibility.<br><br></p>
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| − | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
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| − | <h4 class="panel-title">
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| − | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P1-collapse" aria-expanded="false" aria-controls="P1-collapse">
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| − | <div>
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| − | <div class="col-md-11">Chemical Transformation</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
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| − | </a>
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| − | </h4>
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| − | <p>
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| − | <b>Materials:</b><br>
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| − | LB broth<br>
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| − | Ice<br>
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| − | Selection plates<br>
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| − | <br>
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| − | <b>Methods:</b><br>
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| − | <ol style="font-size:16px;">
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| − | <li>Thaw 50µL competent E. coli cells on ice for 10 minutes<br></li>
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| − | <li>Add:
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| − | <ul style="font-size:16px;">
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| − | <li>5-10 µl DNA from a ligation reaction mix or </li>
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| − | <li>10-100ng DNA of a known plasmid </li>
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| − | </ul>
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| − | </li>
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| − | <li>Carefully flick the tube 4-5 times to mix cells and DNA. <b>Do not vortex.</b></li>
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| − | <li>Place the mixture on ice for 30 minutes. <b>Do not mix.</b></li>
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| − | <li>Heat shock at exactly 42°C for exactly 30 seconds. <b>Do not mix.</b></li>
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| − | <li>Place on ice for 5 minutes. <b>Do not mix.</b></li>
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| − | <li>Pipette 950 µl of room temperature SOC or LB media into the mixture.</li>
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| − | <li>Incubate at 37°C and 200-250 rpm for 60 minutes.</li>
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| − | <li>Mix the cells thoroughly by flicking the tube and inverting.</li>
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| − | <li>Spread:
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| − | <ul style="font-size:16px;">
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| − | <li>For ligation reaction DNA: 100µl of each transformation reaction onto a selection plate. For the rest of 900 µL:
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| − | <ol style="font-size:16px;">
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| − | <li>Pellet cells at 8000rpm for 3 minutes</li>
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| − | <li>Remove and dispense 600 µL of supernatant </li>
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| − | <li>Re-suspend cells by light vortexing</li>
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| − | <li>Plate resuspended cells as above</li>
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| − | </ol></li>
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| − |
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| − | <li>For known plasmid: 10 & 100 µL of each transformation reaction onto a selection plate. For the rest of 890 µL:
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| − | <ol style="font-size:16px;">
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| − | <li>Pellet cells at 8000rpm for 3 minutes</li>
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| − | <li>Remove and dispense 600 µL of supernatant </li>
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| − | <li>Re-suspend cells by light vortexing</li>
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| − | <li>Plate resuspended cells as above</li>
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| − | </ol></li>
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| − | </ul>
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| − | <li>Incubate overnight at 37°C with plates upside down.</li>
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| − | </ol>
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| − | </p>
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| − | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
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| − | <h4 class="panel-title">
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| − | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P2-collapse" aria-expanded="false" aria-controls="P2-collapse">
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| − | <div>
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| − | <div class="col-md-11">Growing Overnight Cultures</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
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| − | </div>
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| − | </a>
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| − | </h4>
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| − | </div>
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| − | <div id="P2-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P2">
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| − | <div class="panel-body">
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| − | <p>
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| − | <b>Materials:</b><br>
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| − | 5 ml LB broth<br>
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| − | 5 μl antibiotic<br>
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| − | Loops<br>
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| − | 12 ml culture tube<br>
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| − | <br>
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| − | <b>Methods:</b><br>
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| − | Overnight cultures were prepared under sterile conditions using a Bunsen burner<br>
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| − | <ol style="font-size:16px;">
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| − | <li>Add 5 ml liquid LB media into 12 ml culture tubes</li>
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| − | <li>Add 5 μl of appropriate antibiotic into the broth</li>
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| − | <li>Using the loop, pick a single colony and inoculate the cultures by dipping the loop into the LB broth</li>
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| − | <li>Seal the tubes and incubate overnight at 37°C shaking at 200-250 rpm</li>
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| − | </ol>
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| − | </p>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | <div class="some-padding"></div>
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| − | <div class="some-padding"></div>
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| | <h2>Paracoccus denitrificans protocols</h2> | | <h2>Paracoccus denitrificans protocols</h2> |
