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| − | <span id="header-title">Materials </span><br>
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| − | <span id="header-subtitle" style="white-space: pre-line;"> & Methods</span>
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| − | <h1>Methods</h1>
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| − | <div class="col-md-11">Growing Overnight Cultures</div><div class="col-md-1"><i class="fa fa-caret-down fa-10" aria-hidden="true"></i></div>
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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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| − | <h4 class="panel-title">
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| − | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P3-collapse" aria-expanded="false" aria-controls="P3-collapse">
| |
| − | <div>
| |
| − | <div class="col-md-11">PCR From Plasmid DNA Template</div><div class="col-md-1"><i class="fa fa-caret-down fa-10" aria-hidden="true"></i></div>
| |
| − | </div>
| |
| − | </a>
| |
| − | </h4>
| |
| − |
| |
| − | </div>
| |
| − | <div id="P3-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P3">
| |
| − | <div class="panel-body">
| |
| − | <p>
| |
| − | <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>
| |
| − | <div class="table-responsive-vertical" style="padding-top: 30px;overflow-x:auto;">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <thead>
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| − | <tr>
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| − | <th>Step</th>
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| − | <th>Temperature (°C)</th>
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| − | <th>Time</th>
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| − |
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| − | </tr>
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| − | </thead>
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <td>Initial denaturation</td>
| |
| − | <td>98</td>
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| − | <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>
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| − | <td>Hold</td>
| |
| − | <td>4</td>
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| − | <td>Indefinitely</td>
| |
| − | </tr>
| |
| − |
| |
| − | </tbody>
| |
| − | </table>
| |
| − | </div>
| |
| − |
| |
| − | <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>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
| |
| − | <div class="panel panel-default">
| |
| − | <div class="panel-heading" role="tab" id="P4">
| |
| − | <h4 class="panel-title">
| |
| − | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P4-collapse" aria-expanded="false" aria-controls="P4-collapse">
| |
| − | <div>
| |
| − | <div class="col-md-11">Colony PCR</div><div class="col-md-1"><i class="fa fa-caret-down fa-10" aria-hidden="true"></i></div>
| |
| − | </div>
| |
| − | </a>
| |
| − | </h4>
| |
| − |
| |
| − | </div>
| |
| − | <div id="P4-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P4">
| |
| − | <div class="panel-body">
| |
| − | <p>
| |
| − | <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>
| |
| − | <div class="table-responsive-vertical" style="padding-top: 30px;overflow-x:auto;">
| |
| − | <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>
| |
| − | </div>
| |
| − | <p>Note: If loading on a gel, the RedTaq mix contains loading dye, so don’t add anything else.</p>
| |
| − |
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | <h1 style="padding-top: 50px !important;">Materials</h1>
| |
| − | <div class="table-responsive-vertical" style="padding-top: 30px;overflow-x:auto;">
| |
| − | <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>
| |
| − | </div>
| |
| − | <h1><span>Marker</span></h1>
| |
| − | <div class="table-responsive-vertical" style="padding-top: 30px;overflow-x:auto;">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <thead>
| |
| − | <tr>
| |
| − | <th>Header</th>
| |
| − | <th>Another Header</th>
| |
| − | </tr>
| |
| − | </thead>
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <td>field 1</td>
| |
| − | <td>value one</td>
| |
| − | </tr>
| |
| − | </tbody>
| |
| − | </table>
| |
| − | </div>
| |
| − | <p><br />
| |
| − | </p>
| |
| − | <h1><span>Enzymes</span></h1>
| |
| − | <div class="table-responsive-vertical" style="padding-top: 30px;overflow-x:auto;">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <thead>
| |
| − | <tr>
| |
| − | <th> Enzyme </th><th> Supplier </th><th> Catalog Number
| |
| − | </th></tr>
| |
| − | </thead>
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <td> DreamTaq Green PCR Master Mix (2X) </td><td> Thermo Fisher Scientific Biosciences GmbH </td><td> K1081
| |
| − | </td></tr>
| |
| − | <tr>
| |
| − | <td> DreamTaq PCR MM </td><td> Fermentas Life Sciences </td><td> K1071
| |
| − | </td></tr>
| |
| − | <tr>
| |
| − | <td> Gibson Assembly® Master Mix </td><td> New England Biolabs </td><td> E2611 S
| |
| − | </td></tr>
| |
| − | <tr>
| |
| − | <td> Lysozyme from Chicken Egg White </td><td> Sigma-Aldrich Chemie GmbH </td><td> L4919-500MG
| |
| − | </td></tr>
| |
| − | <tr>
| |
| − | <td> Phusion® Flash High-Fidelity PCR Master Mix </td><td> Biozym Scientific GmbH </td><td> F-548L
| |
| − | </td></tr>
| |
| − | <tr>
| |
| − | <td> Phusion® High-Fidelity PCR Master Mix </td><td> New England Biolabs </td><td> M0531 L
| |
| − | </td></tr>
| |
| − | <tr>
| |
| − | <td> T4 DNA Ligase </td><td> New England Biolabs GmbH </td><td> M0202 S
| |
| − | </td></tr>
| |
| − | <tr>
| |
| − | <td> 2x PCR Master mix Solution (iTaq) </td><td> HISS DIAGNOSTICS GmbH </td><td> 25028
| |
| − | </td></tr>
| |
| − | <tr>
| |
| − | <td> Sortase A (Staphylococcus aureus) </td><td> roboklon </td><td> E4400-01
| |
| − | </td></tr>
| |
| − | <tr>
| |
| − | <td> TEV Protease </td><td> Th. Geyer GmbH </td><td> SA/T4455/000001
| |
| − | </td></tr>
| |
| − | <tr>
| |
| − | <td> Xylanase, recombinant </td><td> Sigma-Aldrich Chemie GmbH </td><td> X2753-10G
| |
| − | </td></tr>
| |
| − | <tr>
| |
| − | <td> Human DNA (cytosine-5) Methyltransferase </td><td> NEB </td><td> M0230 S
| |
| − | </td></tr></tbody></table>
| |
| − | </div>
| |
| − | </div>
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| − | </article>
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| − | <!-- Saved in parser cache with key 2017_igem_org:pcache:idhash:20811-0!*!*!*!*!*!* and timestamp 20171018070325 and revision id 194737
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| − | -->
| |
| − | </div> <div class="visualClear"></div>
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| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | </div>
| |
| − |
| |
| − | <!-- Side Menubar -->
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| − | <div id="sideMenu">
| |
| − | <a href="https://2017.igem.org">
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| − | <div id="home_logo" >
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| − | <img src="https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg">
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| − | </div>
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| − | </a>
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| − |
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| − | <div style="clear:both; height:5px;"></div>
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| − |
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| − | <div id="menuDisplay"></div> <!- Menu will be loaded here ->
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| − | </div>
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| − |
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| − | <!-- Pop_Why Div is definded here -->
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| − | <div class="pop_why_cover"></div>
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| − |
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| − | <div class="pop_why_box" >
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| − | <div class="pop_close">× </div>
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| − | <div class="pop_why_content"><h3> Loading ... </h3></div>
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| − | </div>
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| − |
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| − | </div>
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| − |
| |
| − | </p>
| |
| − | <!--
| |
| − | NewPP limit report
| |
| − | CPU time usage: 0.006 seconds
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| − | Real time usage: 0.006 seconds
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| − | Preprocessor visited node count: 4/1000000
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| − | Preprocessor generated node count: 24/1000000
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| − | Post‐expand include size: 0/2097152 bytes
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| − | Template argument size: 0/2097152 bytes
| |
| − | Highest expansion depth: 2/40
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| − | Expensive parser function count: 0/100
| |
| − | -->
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| − |
| |
| − | <!-- Saved in parser cache with key 2017_igem_org:pcache:idhash:20811-0!*!*!*!*!*!* and timestamp 20171018080953 and revision id 194836
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| − | -->
| |
| − | </div> <div class="visualClear"></div>
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| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | </div>
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| − |
| |
| − | <!-- Side Menubar -->
| |
| − | <div id="sideMenu">
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| − | <a href="https://2017.igem.org">
| |
| − | <div id="home_logo" >
| |
| − | <img src="https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg">
| |
| − | </div>
| |
| − | </a>
| |
| − |
| |
| − | <div style="clear:both; height:5px;"></div>
| |
| − |
| |
| − | <div id="menuDisplay"></div> <!- Menu will be loaded here ->
| |
| − | </div>
| |
| − |
| |
| − | <!-- Pop_Why Div is definded here -->
| |
| − | <div class="pop_why_cover"></div>
| |
| − |
| |
| − | <div class="pop_why_box" >
| |
| − | <div class="pop_close">× </div>
| |
| − | <div class="pop_why_content"><h3> Loading ... </h3></div>
| |
| − | </div>
| |
| − |
| |
| − | </div>
| |
| − | </html>
| |
