|
|
| Line 35: |
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| | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P2-collapse" aria-expanded="false" aria-controls="P2-collapse"> | | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P2-collapse" aria-expanded="false" aria-controls="P2-collapse"> |
| | <div> | | <div> |
| − | <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> | + | <div class="col-md-11"> Chemically Competent Cells</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> |
| | </div> | | </div> |
| | </a> | | </a> |
| Line 43: |
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| | <div id="P2-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P2"> | | <div id="P2-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P2"> |
| | <div class="panel-body"> | | <div class="panel-body"> |
| − | <p> | + | <img src="https://static.igem.org/mediawiki/2018/b/bf/T--East_Chapel_Hill--GELEXTRACT.png" |
| − | <b>Materials:</b><br>
| + | |
| − | 5 ml LB broth<br>
| + | |
| − | 5 μl antibiotic<br>
| + | |
| − | Loops<br>
| + | |
| − | 12 ml culture tube<br>
| + | |
| − | <br>
| + | |
| − | <b>Methods:</b><br>
| + | |
| − | Overnight cultures were prepared under sterile conditions using a Bunsen burner<br>
| + | |
| − | <ol style="font-size:16px;">
| + | |
| − | <li>Add 5 ml liquid LB media into 12 ml culture tubes</li>
| + | |
| − | <li>Add 5 μl of appropriate antibiotic into the broth</li>
| + | |
| − | <li>Using the loop, pick a single colony and inoculate the cultures by dipping the loop into the LB broth</li>
| + | |
| − | <li>Seal the tubes and incubate overnight at 37°C shaking at 200-250 rpm</li>
| + | |
| − | </ol>
| + | |
| − | </p>
| + | |
| − | | + | |
| | </div> | | </div> |
| | </div> | | </div> |
| Line 81: |
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| | <div id="P3-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P3"> | | <div id="P3-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P3"> |
| | <div class="panel-body"> | | <div class="panel-body"> |
| − | <p> | + | <img src="https://static.igem.org/mediawiki/2018/b/bf/T--East_Chapel_Hill--GELEXTRACT.png" |
| − | <b>Materials:</b><br>
| + | |
| − | 2x Phusion Mastermix<br>
| + | |
| − | 10 µM forward primer<br>
| + | |
| − | 10 µM forward primer<br>
| + | |
| − | PCR tube<br>
| + | |
| − | Sterile water<br>
| + | |
| − | Plasmid DNA<br>
| + | |
| − | <br>
| + | |
| − | <b>Methods:</b><br>
| + | |
| − | For a 25 µL reaction<br>
| + | |
| − | <ol style="font-size:16px;">
| + | |
| − | <li>In a PCR tube on ice, combine 1-10 ng of plasmid DNA, 1.25 µL of 10 µM forward primer, 1.25 µL of 10 µM reverse primer to a PCR tube on ice, 12.5 µL of 2x Phusion Mastermix, and sterile water up to 25 µL.
| + | |
| − | <br><i>Note: It is important to add Phusion Master Mix last in order to prevent primer degradation
| + | |
| − | caused by the 3 ́→ 5 ́ exonuclease activity</i></li>
| + | |
| − | <li>Gently mix the reaction</li>
| + | |
| − | <li>If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly</li>
| + | |
| − | <li>Transfer the PCR tube from ice to a PCR machine to begin thermocycling</li>
| + | |
| − | </ol>
| + | |
| − | | + | |
| − | <p><br>For a 50 µL reaction<br></p>
| + | |
| − | <ol style="font-size:16px;">
| + | |
| − | <li>In a PCR tube on ice, combine 1-10 ng of plasmid DNA, 2.50 µL of 10 µM forward primer, 2.50 µL of 10 µM reverse primer to a PCR tube on ice, 25 µL of 2x Phusion Mastermix, and sterile water up to 50 µL.
| + | |
| − | <br><i>Note: It is important to add Phusion Master Mix last in order to prevent primer degradation
| + | |
| − | caused by the 3 ́→ 5 ́ exonuclease activity</i></li>
| + | |
| − | <li>Gently mix the reaction</li>
| + | |
| − | <li>If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly</li>
| + | |
| − | <li>Transfer the PCR tube from ice to a PCR machine preheated to 98°C to begin thermocycling</li>
| + | |
| − | </ol>
| + | |
| − | <p><br><b>Thermocycling</b><br>
| + | |
| − | The PCR machine should be set to run the following steps: </p>
| + | |
| − | <table class="table table-bordered table-striped">
| + | |
| − | <thead>
| + | |
| − | <tr>
| + | |
| − | <th>Step</th>
| + | |
| − | <th>Temperature (°C)</th>
| + | |
| − | <th>Time</th>
| + | |
| − |
| + | |
| − | </tr>
| + | |
| − | </thead>
| + | |
| − | <tbody>
| + | |
| − | <tr>
| + | |
| − | <td>Initial denaturation</td>
| + | |
| − | <td>98</td>
| + | |
| − | <td>30 seconds</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>25-35 cycles</td>
| + | |
| − | <td>98 (denaturation)<br>
| + | |
| − | 45-72 (annealing) <a href="#Note1">see Note 1</a><br>
| + | |
| − | 72 (extension)</td>
| + | |
| − | <td>5-10 seconds <br>
| + | |
| − | 10-30 seconds<br>
| + | |
| − | 15-30 seconds per kb</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Final extension</td>
| + | |
| − | <td>72</td>
| + | |
| − | <td>2-5 minutes</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Hold</td>
| + | |
| − | <td>4</td>
| + | |
| − | <td>Indefinitely</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | </tbody>
| + | |
| − | </table>
| + | |
| − | | + | |
| − | <p id="Note1">Note 1: Use the NEB Tm calculator should be used to determine the annealing temperature when using Phusion: <a target="_blank" href="http://tmcalculator.neb.com/#!/">http://tmcalculator.neb.com/#!/</a></p>
| + | |
| − | | + | |
| − | | + | |
| − | </p>
| + | |
| | | | |
| | </div> | | </div> |
| Line 176: |
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| | <div id="P4-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P4"> | | <div id="P4-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P4"> |
| | <div class="panel-body"> | | <div class="panel-body"> |
| − | <p> | + | <img src="https://static.igem.org/mediawiki/2018/b/bf/T--East_Chapel_Hill--GELEXTRACT.png" |
| − | <b>Materials:</b><br>
| + | |
| − | Sterile Water<br>
| + | |
| − | 25 µL RedTaq mastermix<br>
| + | |
| − | 1 E. coli colony<br>
| + | |
| − | 2.5 µL of 10 µM forward primer<br>
| + | |
| − | 2.5 µL of 10 µM reverse primer<br>
| + | |
| − | <br>
| + | |
| − | <b>Methods:</b><br>
| + | |
| − | <ol style="font-size:16px;">
| + | |
| − | <li>Add a single colony of cells to 50 µL of water. Incubate at 95C for a minute to lyse the cells.</li>
| + | |
| − | <li>Combine 1 µL cell lysate, 25 µL RedTaq mastermix, 2.5 µL of 10 µM forward primer, 2.5 µL of 10 µM reverse primer, and sterile water up to 50 µL. <br>
| + | |
| − | <i>Note: It is important to add RedTaq Master Mix last in order to prevent primer
| + | |
| − | degradation caused by the 3 ́→ 5 ́ exonuclease activity</i></li>
| + | |
| − | <li>Incubate in the thermocycler - Taq has a lower optimum temperature than Phusion.</li>
| + | |
| − | | + | |
| − | </ol>
| + | |
| − | | + | |
| − | <p><br><b>Thermocycling</b><br>
| + | |
| − | The PCR machine should be set to run the following steps: </p>
| + | |
| − | <table class="table table-bordered table-striped">
| + | |
| − | <thead>
| + | |
| − | <tr>
| + | |
| − | <th>Step</th>
| + | |
| − | <th>Temperature (°C)</th>
| + | |
| − | <th>Time</th>
| + | |
| − |
| + | |
| − | </tr>
| + | |
| − | </thead>
| + | |
| − | <tbody>
| + | |
| − | <tr>
| + | |
| − | <td>Initial denaturation</td>
| + | |
| − | <td>98</td>
| + | |
| − | <td>30 seconds</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>25-35 cycles</td>
| + | |
| − | <td>98 (denaturation)<br>
| + | |
| − | 45-72 (annealing) <a href="#Note1">see Note 1</a><br>
| + | |
| − | 68 (extension)</td>
| + | |
| − | <td>5-10 seconds <br>
| + | |
| − | 10-30 seconds<br>
| + | |
| − | 15-30 seconds per kb</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Final extension</td>
| + | |
| − | <td>72</td>
| + | |
| − | <td>5-10 minutes</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Hold</td>
| + | |
| − | <td>4</td>
| + | |
| − | <td>Indefinitely</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | </tbody>
| + | |
| − | </table>
| + | |
| − | <p>Note: If loading on a gel, the RedTaq mix contains loading dye, so don’t add anything else.</p>
| + | |
| − | | + | |
| | </div> | | </div> |
| | </div> | | </div> |
| Line 257: |
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| | <div class="panel-body"> | | <div class="panel-body"> |
| | | | |
| − | <p> | + | <img src="https://static.igem.org/mediawiki/2018/b/bf/T--East_Chapel_Hill--GELEXTRACT.png" |
| − | <specialh3>LB Broth</specialh3><br>
| + | |
| − | <b>Materials:</b><br>
| + | |
| − | 25 g LB broth (powder)<br>
| + | |
| − | 1 Litre Purified Water<br>
| + | |
| − | <br>
| + | |
| − | <b>Methods:</b><br>
| + | |
| − | <ol style="font-size:16px;">
| + | |
| − | <li>Add 25g LB broth to 1 litre purified water</li>
| + | |
| − | <li>Autoclave</li>
| + | |
| − | </ol>
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p>
| + | |
| − | <specialh3>LB Agar</specialh3><br>
| + | |
| − | <b>Materials:</b><br>
| + | |
| − | 37 g LB Agar (powder)<br>
| + | |
| − | 1 Litre Purified Water<br>
| + | |
| − | <br>
| + | |
| − | <b>Methods:</b><br>
| + | |
| − | <ol style="font-size:16px;">
| + | |
| − | <li>Add 37g LB Agar to 1 litre purified water</li>
| + | |
| − | <li>Autoclave</li>
| + | |
| − | </ol>
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p>
| + | |
| − | <specialh3>Glycerol Stocks</specialh3><br>
| + | |
| − | <b>Materials:</b><br>
| + | |
| − | 500µl glycerol (80%)<br>
| + | |
| − | 500µl overnight culture in LB<br>
| + | |
| − | <br>
| + | |
| − | <b>Methods:</b><br>
| + | |
| − | <ol style="font-size:16px;">
| + | |
| − | <li>Add 500µl glycerol (80%) to 1.5ml eppendorf tube</li>
| + | |
| − | <li>Add 500µl overnight culture in LB</li>
| + | |
| − | <li>Store at -80°C</li>
| + | |
| − | </ol>
| + | |
| − | </p>
| + | |
| | </div> | | </div> |
| | </div> | | </div> |
| Line 317: |
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| | <div id="P6-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P6"> | | <div id="P6-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P6"> |
| | <div class="panel-body"> | | <div class="panel-body"> |
| − | <p> | + | <img src="https://static.igem.org/mediawiki/2018/b/bf/T--East_Chapel_Hill--GELEXTRACT.png" |
| − | <b>Materials:</b><br>
| + | |
| − | Agarose Powder<br>
| + | |
| − | TAE buffer<br>
| + | |
| − | Gel mould<br>
| + | |
| − | 5-10 µL SybrSafe<br>
| + | |
| − | Gel Tank<br>
| + | |
| − | 8-10 µL DNA ladder<br>
| + | |
| − | DNA loading dye<br>
| + | |
| − | <br>
| + | |
| − | <b>Methods:</b><br>
| + | |
| − | <ol style="font-size:16px;">
| + | |
| − | <li>Prepare 8% w/v solution of agarose powder in 1/10 TAE buffer (e.g. 0.8g agarose powder in 100 mL buffer) using a conical flask</li>
| + | |
| − | <li>Heat the mixture until agarose is completely dissolved. Do not let the solution boil.</li>
| + | |
| − | <li>Pour the solution into a gel mould</li>
| + | |
| − | <li>Add 5-10 µL SybrSafe® to the solution. Make sure there are no bubbles in the solution.</li>
| + | |
| − | <li>Allows the solution to set (approx 15-20 minutes)</li>
| + | |
| − | <li>Transfer the agarose gel to a tank, remove the comb and apply:
| + | |
| − | <ul>
| + | |
| − | <li>8-10 µL of the DNA ladder </li>
| + | |
| − | <li>DNA samples with the corresponding amount of DNA loading dye (6X)</li>
| + | |
| − | </ul></li>
| + | |
| − | <li>Run the gel for 45-60 minutes at 100V</li>
| + | |
| − | | + | |
| − | </ol>
| + | |
| − | | + | |
| | | | |
| | </div> | | </div> |
| Line 365: |
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| | <div id="P7-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P7"> | | <div id="P7-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P7"> |
| | <div class="panel-body"> | | <div class="panel-body"> |
| − | <p> | + | <img src="https://static.igem.org/mediawiki/2018/b/bf/T--East_Chapel_Hill--GELEXTRACT.png" |
| − | <b>Materials:</b><br>
| + | |
| − | Microcentrifuge tube<br>
| + | |
| − | Ice<br>
| + | |
| − | 1 µL T4 DNA Ligase<br>
| + | |
| − | 2 µL 10X T4 DNA ligase buffer<br>
| + | |
| − | 50ng Vector Plasmid<br>
| + | |
| − | Insert DNA<br>
| + | |
| − | Sterile water<br>
| + | |
| − | <br>
| + | |
| − | <b>Methods:</b><br>
| + | |
| − | <ol style="font-size:16px;">
| + | |
| − | <li>Calculate volumes of vector and insert DNA required using NEBioCalculator (<a href="http://nebiocalculator.neb.com/#!/ligation" > http://nebiocalculator.neb.com/#!/ligation</a> - required molar ratio of 1:3::vector:insert)</li>
| + | |
| − | <li>Add vector plasmid, insert DNA, T4 DNA ligase and T4 DNA ligase buffer to the microcentrifuge tube on ice (add T4 DNA ligase last)</li>
| + | |
| − | <li>Make reaction up to 20 µL using sterile water</li>
| + | |
| − | <li>Incubate at room temperature for 30 - 60 min for sticky ends or 1-2 hours for blunt ends</li>
| + | |
| − | </ol>
| + | |
| | | | |
| | </div> | | </div> |
| Line 405: |
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| | <div id="P8-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P8"> | | <div id="P8-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P8"> |
| | <div class="panel-body"> | | <div class="panel-body"> |
| − | <p> | + | <img src="https://static.igem.org/mediawiki/2018/b/bf/T--East_Chapel_Hill--GELEXTRACT.png" |
| − | <b>Materials:</b><br>
| + | |
| − | Restriction Enzyme:
| + | |
| − | <a href"https://www.neb.com/tools-and-resources/interactive-tools/enzyme-finder?searchType=7&recognitionSite=&matchType=1">NEB enzyme finder</a> used to see which restriction enzymes are required <br>
| + | |
| − | 10X buffer: <a href="https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder">NEB double digest finder</a> used to see which buffers are required for the particular restriction enzymes <br>
| + | |
| − | Plasmid DNA<br>
| + | |
| − | Sterile water<br>
| + | |
| − | <br>
| + | |
| − | <b>Methods:</b><br>
| + | |
| − | | + | |
| − | <table class="table table-bordered table-striped">
| + | |
| − | <thead>
| + | |
| − | <tr>
| + | |
| − | <th>Component</th>
| + | |
| − | <th>Test digest<br>
| + | |
| − | Double digestion (20 µL, for construct analysis)
| + | |
| − | </th>
| + | |
| − | <th>Assemble digest
| + | |
| − | <br>Double digestion (20-50 µL, for gene assembly)</th>
| + | |
| − |
| + | |
| − | </tr>
| + | |
| − | </thead>
| + | |
| − | <tbody>
| + | |
| − | <tr>
| + | |
| − | <td>Sterile water</td>
| + | |
| − | <td>to 20 µL</td>
| + | |
| − | <td>to 50 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>10X buffer</td>
| + | |
| − | <td>2 µL</td>
| + | |
| − | <td>2-5 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Plasmid DNA</td>
| + | |
| − | <td>~200 ng for test digest,
| + | |
| − | (DNA mass is variable dependent on insert size.<br> Smallest digestion fragment mass should be > 50ng)</td>
| + | |
| − | <td>1,000 -2,000 ng</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Restriction enzyme</td>
| + | |
| − | <td>0.5 µL + 0.5 µL</td>
| + | |
| − | <td>1 µL + 1 µL</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | </tbody>
| + | |
| − | </table>
| + | |
| − | <ol style="font-size:16px;">
| + | |
| − | <li>Set up the reaction following the instruction in the table above , depending on whether test digest or assembly digest is being performed.</li>
| + | |
| − | <li> Incubate digestion reaction at 37°C:
| + | |
| − | <ol>
| + | |
| − | <li>30 min for Test digest</li>
| + | |
| − | <li>2-3 hours or overnight for Assembly digest</li>
| + | |
| − | </ol>
| + | |
| − | </li>
| + | |
| − | <li>Perform heat deactivation at 80°C for 20 minutes, if not running on a gel at the end of incubation. </li>
| + | |
| − | </ol>
| + | |
| | | | |
| | </div> | | </div> |
| Line 484: |
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| | <div id="P9-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P9"> | | <div id="P9-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P9"> |
| | <div class="panel-body"> | | <div class="panel-body"> |
| − | <p> | + | <img src="https://static.igem.org/mediawiki/2018/b/bf/T--East_Chapel_Hill--GELEXTRACT.png" |
| − | <b>Materials:</b><br>
| + | |
| − | Microcentrifuge tube<br>
| + | |
| − | Media of choice<br>
| + | |
| − | Overnight Culture of Cells<br>
| + | |
| − | 96 well plate<br>
| + | |
| − | Plate reader<br>
| + | |
| − | <br>
| + | |
| − | <b>Methods:</b><br>
| + | |
| − | <ol style="font-size:16px;">
| + | |
| − | <li>Dilute overnight cultures to 0.1 OD. <b>Don’t forget the media control.</b></li>
| + | |
| − | <li>Seed your cells in the 96 well plate at 100μL per well.</li>
| + | |
| − | <li>Make reaction up to 20 µL using sterile water</li>
| + | |
| − | <li>Set the plate reader for 600OD and run it for 12 hours for bacteria or 24 hours plus for yeast
| + | |
| − | </li>
| + | |
| − | </ol>
| + | |
| − | | + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="some-padding"></div>
| + | |
| − | <div class="some-padding"></div>
| + | |
| − | | + | |
| − | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
| + | |
| − | <div class="panel panel-default">
| + | |
| − | <div class="panel-heading" role="tab" id="P10">
| + | |
| − | <h4 class="panel-title">
| + | |
| − | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P10-collapse" aria-expanded="false" aria-controls="P10-collapse">
| + | |
| − | <div>
| + | |
| − | <div class="col-md-11">Cell Sorting for Co-Culture Characterisation</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
| + | |
| − | </div>
| + | |
| − | </a>
| + | |
| − | </h4>
| + | |
| − | | + | |
| − | </div>
| + | |
| − | <div id="P10-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P10">
| + | |
| − | <div class="panel-body">
| + | |
| − | <p>
| + | |
| − | <b>Materials:</b><br>
| + | |
| − | Microcentrifuge tube<br>
| + | |
| − | Media of choice<br>
| + | |
| − | Overnight Culture of Cells<br>
| + | |
| − | 96 well plate<br>
| + | |
| − | Flow Cytometer<br>
| + | |
| − | PBS<br>
| + | |
| − | <br>
| + | |
| − | <b>Methods:</b><br>
| + | |
| − | <ol style="font-size:16px;">
| + | |
| − | <li>Use Flowjo to predefine the gates for the Flow cytometer. This allows the cells within the co culture to be counted based on size.</li>
| + | |
| − | <li>Dilute the Cultures to 1 OD (don’t forget the media control) and culture together at desired inoculation ratio</li>
| + | |
| − | <li>Set up samples in triplicate form on a 96 well plate. Dilute 10 fold and 100 fold in PBS.</li>
| + | |
| − | <li>Incubate Cells and take samples at desired time points to obtain growth curves</li>
| + | |
| − | </ol>
| + | |
| | | | |
| | </div> | | </div> |
| Line 561: |
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| | <div id="P11-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P11"> | | <div id="P11-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P11"> |
| | <div class="panel-body"> | | <div class="panel-body"> |
| − | <p> | + | <img src="https://static.igem.org/mediawiki/2018/b/bf/T--East_Chapel_Hill--GELEXTRACT.png" |
| − | <b>Materials:</b><br>
| + | |
| − | Replica plate or glycerol stock of cells<br>
| + | |
| − | Appropriate antibiotic<br>
| + | |
| − | LB broth<br>
| + | |
| − | 96 well plate<br>
| + | |
| − | AHLs (at concentrations of 10pM, 100pM, 1nM, 10nM, 100nM, 1µM, 10µM and 100µM)<br>
| + | |
| − | <br>
| + | |
| − | <b>Methods:</b><br>
| + | |
| − | <ol style="font-size:16px;">
| + | |
| − | <li>Grow overnight culture of cells, either from replica plate or glycerol stock (see overnight culture protocol)
| + | |
| − | </li>
| + | |
| − | <li>In the morning, add 100 µL of the cell suspension to 10 mL LB and 10µL of antibiotic</li>
| + | |
| − | <li>Grow cells in incubator for 3-4 hours at 200rpm and 37°C</li>
| + | |
| − | <li>Blank spectrophotometer with 1mL LB and measure OD of cells (dilute 10x in LB)</li>
| + | |
| − | <li>Make up suspension of cells at OD 0.05</li>
| + | |
| − | <li>Add 100µL of cells to each well in the 96-well plate using the block-filler machine</li>
| + | |
| − | <li>Using the multi-channel pipette add 2µL of intended AHL to each well, add samples in triplicate form
| + | |
| − | </li>
| + | |
| − | <li>Using the multi-channel pipette add 2µL of intended AHL to each well, add samples in triplicate form
| + | |
| − | </li>Run on plate-reader for 12 hours, with fluorescence and absorbance measurements taken every 15 minutes (set plate reader to 37°C and 500rpm shaking)
| + | |
| − | </li>
| + | |
| − | </ol>
| + | |
| − | | + | |
| | </div> | | </div> |
| | </div> | | </div> |
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| | <div id="P12-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P12"> | | <div id="P12-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P12"> |
| | <div class="panel-body"> | | <div class="panel-body"> |
| − | <p> | + | <img src="https://static.igem.org/mediawiki/2018/b/bf/T--East_Chapel_Hill--GELEXTRACT.png" |
| − | <b>PCR purification</b> was performed according to the QIAquick PCR Purification Kit<br>
| + | |
| − | <b>Gel extraction</b> was performed according to the QIAquick Gel Extraction Kit<br>
| + | |
| − | <b>Minipreps</b> were carried out according to the QIAprep Spin Miniprep Kit<br>
| + | |
| − | | + | |
| − | | + | |
| | </div> | | </div> |
| | </div> | | </div> |
