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| | <html> | | <html> |
| | <style> | | <style> |
| − | .body {
| + | .myintro { |
| − | margin: 20%; }
| + | |
| − | | + | |
| − | .table {
| + | |
| − | width: 50%;
| + | |
| − | text-align: center;
| + | |
| − | margin-left: 25% !important;
| + | |
| − | margin-right: 25%;
| + | |
| − | }
| + | |
| − | | + | |
| − | .myintro {
| + | |
| | display: table; | | display: table; |
| | width: 100%; | | width: 100%; |
| Line 20: |
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| | text-align: center; | | text-align: center; |
| | color: black; | | color: black; |
| − | background: url("https://static.igem.org/mediawiki/2017/3/3a/T--BOKU-Vienna--Notebook_Backgroundgrey.jpg") no-repeat top center scroll; | + | background: url("https://static.igem.org/mediawiki/2017/b/b6/T--BOKU-Vienna--dna.jpg") no-repeat bottom center scroll; |
| | background-color: black; | | background-color: black; |
| | -webkit-background-size: cover; | | -webkit-background-size: cover; |
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| | font-family: Tahoma, Geneva, sans-serif; | | font-family: Tahoma, Geneva, sans-serif; |
| | } | | } |
| − | | + | .invert { |
| − | .newstyle { | + | -webkit-filter: invert(1); |
| − | vertical-align: left;
| + | filter: invert(1) brightness(150%); |
| − | color: white
| + | } |
| − | }
| + | |
| − | | + | |
| − | .table {
| + | |
| − | color: white;
| + | |
| − | }
| + | |
| − | | + | |
| − | .mylist {
| + | |
| − | text-align: left;
| + | |
| − | color: white !important;
| + | |
| − | }
| + | |
| − |
| + | |
| − | .mydiv {
| + | |
| − | float:none;
| + | |
| − | display:inline-block;
| + | |
| − | *display:inline; /* ie7 fix */
| + | |
| − | *zoom:1; /* hasLayout ie7 trigger */
| + | |
| − | vertical-align: top;
| + | |
| − | }
| + | |
| − | | + | |
| − | .small {
| + | |
| − | font-size:90%;
| + | |
| − | }
| + | |
| − | | + | |
| − | .myanchor {
| + | |
| − | display: block;
| + | |
| − | position: absolute;
| + | |
| − | width: 0;
| + | |
| − | height: 0;
| + | |
| − | z-index: -1;
| + | |
| − | top: -40px;
| + | |
| − | left: 0;
| + | |
| − | visibility: hidden;
| + | |
| − | }
| + | |
| − | | + | |
| − | | + | |
| − | #materials {
| + | |
| − | /* places the contents of the element 100px from the top of the view-port */
| + | |
| − | padding-top: 100px;
| + | |
| − | }
| + | |
| − | | + | |
| − | #lbmedium1 {
| + | |
| − | /* places the contents of the element 100px from the top of the view-port */
| + | |
| − | padding-top: 100px;
| + | |
| − | }
| + | |
| − | | + | |
| − | #lbagar {
| + | |
| − | padding-top: 100px;
| + | |
| − | }
| + | |
| − | | + | |
| − | #methods {
| + | |
| − | /* places the contents of the element 100px from the top of the view-port */
| + | |
| − | padding-top: 100px;
| + | |
| − | }
| + | |
| − | | + | |
| − | #miniprep {
| + | |
| − | /* places the contents of the element 100px from the top of the view-port */
| + | |
| − | padding-top: 100px;
| + | |
| − | }
| + | |
| − | | + | |
| − | #transformation {
| + | |
| − | /* places the contents of the element 100px from the top of the view-port */
| + | |
| − | padding-top: 100px;
| + | |
| − | }
| + | |
| − | | + | |
| − | #transformationyeast {
| + | |
| − | /* places the contents of the element 100px from the top of the view-port */
| + | |
| − | padding-top: 100px;
| + | |
| − | }
| + | |
| − | | + | |
| − | #interlab {
| + | |
| − | padding-top: 100px;
| + | |
| − | }
| + | |
| − | | + | |
| − | #q5pcr {
| + | |
| − | padding-top: 100px;
| + | |
| − | }
| + | |
| − | | + | |
| − | #goldengate {
| + | |
| − | padding-top: 100px;
| + | |
| − | }
| + | |
| − | | + | |
| − | #cpcr {
| + | |
| − | padding-top: 100px;
| + | |
| − | }
| + | |
| − | | + | |
| | </style> | | </style> |
| | | | |
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| | | | |
| | <!-- About Section --> | | <!-- About Section --> |
| − | <section id="Protocol" class="container content-section text-center"> | + | |
| − | <div class="row"> | + | <section id="Protocol" class="container content-section text-center Protocol"> |
| | + | <div class="container text-center content-section Protocol"> |
| | <div class="col-lg-8 col-lg-offset-2"> | | <div class="col-lg-8 col-lg-offset-2"> |
| − | <h2>Protocols from A-Z.</h2> | + | <h2>Protocols?</h2> |
| − | <p> <br> Our protocols will follow shortly. </br> | + | <p>Under construction...</p> |
| − | </p>
| + | |
| | + | |
| | | | |
| | </div> | | </div> |
| | </div> | | </div> |
| | + | </section> |
| | | | |
| − | <div class="mydiv">
| |
| − | <h3>Contents</h3>
| |
| − | <p></p>
| |
| − | <p></p>
| |
| − | <ul class="mylist">
| |
| − | <li><a href="#materials">Materials</a>
| |
| − | <ul>
| |
| − | <li class="small"><a href="#lbmedium1">LB medium - E. Coli</a></li>
| |
| − | </ul>
| |
| − | <ul>
| |
| − | <li class="small"><a href="#lbagar">LB Agar - E. Coli</a></li>
| |
| − | </ul>
| |
| − | <li><a href="#methods">Methods</a>
| |
| − | <ul>
| |
| − | <li class="small"><a href="#miniprep">Miniprep</a></li>
| |
| − | </ul>
| |
| − | <ul>
| |
| − | <li class="small"><a href="#transformation">Transformation of chemically competent E. Coli</a></li>
| |
| − | </ul>
| |
| − | <ul>
| |
| − | <li class="small"><a href="#transformationyeast">Preparation of electro-competent Pichia pastoris cells</a></li>
| |
| − | </ul>
| |
| − | <ul>
| |
| − | <li class="small"><a href="#interlab">InterLab Study</a></li>
| |
| − | </ul>
| |
| − | <ul>
| |
| − | <li class="small"><a href="#q5pcr">Q5 PCR</a></li>
| |
| − | </ul>
| |
| − | <ul>
| |
| − | <li class="small"><a href="#goldengate">Golden Gate</a></li>
| |
| − | </ul>
| |
| − | <ul>
| |
| − | <li class="small"><a href="#cpcr">OneTaq Colony PCR</a></li>
| |
| − | </ul>
| |
| − |
| |
| − |
| |
| − | </ul>
| |
| | | | |
| − | </div>
| |
| | | | |
| − | <div>
| |
| − | <h3 id="materials">Materials</h3>
| |
| − |
| |
| − |
| |
| − | <h4 id="lbmedium1">LB medium - E. Coli</h4>
| |
| − |
| |
| − | <table class="table">
| |
| − | <thead>
| |
| − | <tr>
| |
| − | <th>Name</th>
| |
| − | <th>Amount</th>
| |
| − | </tr>
| |
| − | </thead>
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <td>Hy Soy Peptone</td>
| |
| − | <td>10.0g</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Yeast Extract</td>
| |
| − | <td>5.0g</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>NaCl</td>
| |
| − | <td>5.0g</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>RO-water</td>
| |
| − | <td>1000 mL</td>
| |
| − | </tr>
| |
| − | </tbody>
| |
| − | </table>
| |
| − | </div>
| |
| − | <div>
| |
| − | <h4 id="lbagar">LB Agar - E. Coli</h4>
| |
| − |
| |
| − | <table class="table">
| |
| − | <thead>
| |
| − | <tr>
| |
| − | <th>Name</th>
| |
| − | <th>Amount</th>
| |
| − | </tr>
| |
| − | </thead>
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <td>Hy soy Peptone</td>
| |
| − | <td>10.0g</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Yeast Extract</td>
| |
| − | <td>5.0g</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>NaCl</td>
| |
| − | <td>5.0g</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>RO-water</td>
| |
| − | <td>980 mL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Agar-Agar per 500mL</td>
| |
| − | <td>7g</td>
| |
| − | </tr>
| |
| − | </tbody>
| |
| − | </table>
| |
| − | </div>
| |
| − |
| |
| − | <div>
| |
| − | <h3 id="methods">Methods</h3>
| |
| − | <h4 id="transformation">Transformation of chemically competent E. Coli</h4>
| |
| − | <p></p>
| |
| − | <p> Competent cells from E. Coli strain DH5α or DH10B were thawed on ice. The whole ligation/other DNA samples were added to the cells, for 30 min the mix was put on ice. The cells and DNA mix were heat shocked for 90s at 42°C. The cells recovered on ice for 5min. 1 mL of LB medium was added to the cells and then an incubation followed at 37°C in a shaker-incubator for 45min. An appropiate amount of cells were plated on a selective LB agar.</p>
| |
| − |
| |
| − | <h4 id="transformationyeast">Preparation of electro-competent Pichia pastoris cells</h4>
| |
| − | <p></p>
| |
| − | <p>Pichia pastoris cells are generated, which are competent for transformation by electroporation. For the pre-culture, inoculate a selective YPD medium and incubate over night on a shaker. For the main culture, inoculate non-selective YPD medium with the pre-culture and incubate again over night. On the next day, measure the OD and split the culture into two falcon tubes. Centrifuge the cells and discard the supernatant. Add pre-treating solution to each of the pellets,
| |
| − | resuspend and incubate the cells for 30 minutes. Add ice-cold sorbitol to both Falcon-tubes and harvest cells, then discard the supernatant. Combine the pellets of the two tubes and resuspend in ice-cold sorbitol. Harvest them and discard the supernatant. Resuspend the cells in sorbitol and store it in Eppendorf tubes on ice until transformation. For electoporation, an aliquot of the cells is mixed with linearized DNA. As negative control, cells are transformed with elution solution or AD. Transfer the mix into a cooled electroporation cuvette and incubate it on ice for 5 minutes. Set the parameters on the electroporator to 2000 V, 25µF and 186 Ω. After transformation 1 mL of YPD-media is added to the cells in the cuvette, then they are transferred into a microcentrifuge tube. Regenerate the cells for 1.5h-3h at 28°C. Then plate some of the cells onto selective agar plates and incubate at 28°C until colonies appear. The rest is kept on agar plates at 4°C.
| |
| − | </p>
| |
| − |
| |
| − | <h4 id="miniprep">Miniprep</h4>
| |
| − | <p></p>
| |
| − | <p>Minipreps were performed using Hi Yield®Plasmid Mini DNA Isolationkit according to the manufacturer's protocols.
| |
| − | </p>
| |
| − |
| |
| − | <h4 id="interlab">InterLab Study</h4>
| |
| − | <p></p>
| |
| − | <p>The Interlab Challenge was performed according to the protocols for the InterLab Study in 2017. For further details visit the iGEM homepage: <a href="https://2017.igem.org/Competition/InterLab_Study" target="_blank">InterLab Study</a> or our <a href=" https://2017.igem.org/Team:BOKU-Vienna/InterLab" target="_blank">InterLab page</a>.</p>
| |
| − | <h5>Kit Plate 6 InterLab Part Locations</h5>
| |
| − | <table class="table">
| |
| − | <thead>
| |
| − | <tr>
| |
| − | <th>Name</th>
| |
| − | <th>Backbone</th>
| |
| − | </tr>
| |
| − | </thead>
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <td>Positive Control</td>
| |
| − | <td>BBa_I20270</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Negative Control</td>
| |
| − | <td>BBa_R0040</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Test Device 1</td>
| |
| − | <td>BBa_J364000</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Test Device 2</td>
| |
| − | <td>BBa_J364001</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Test Device 3</td>
| |
| − | <td>BBa_J364002</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Test Device 4</td>
| |
| − | <td>BBa_J364003</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Test Device 5</td>
| |
| − | <td>BBa_J364004</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Test Device 6</td>
| |
| − | <td>BBa_J364005</td>
| |
| − | </tr>
| |
| − | </tbody>
| |
| − | </table>
| |
| − |
| |
| − | <h4 id="q5pcr">Q5 PCR</h4>
| |
| − | <p></p>
| |
| − | <table class="table">
| |
| − | <thead>
| |
| − | <tr>
| |
| − | <th>Name</th>
| |
| − | <th>Amount</th>
| |
| − | </tr>
| |
| − | </thead>
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <td>in PCR tube aliquoted oneTaq 2x MM</td>
| |
| − | <td>12.5 µL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>AD</td>
| |
| − | <td>variable</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Primer forward</td>
| |
| − | <td>1.25 µL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Primer reverse</td>
| |
| − | <td>1.25 µL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Template</td>
| |
| − | <td>1 ng</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Total Volume</td>
| |
| − | <td>25 µL</td>
| |
| − | </tr>
| |
| − |
| |
| − | </tbody>
| |
| − | </table>
| |
| − |
| |
| − | <p>Vortex and spin down PCR tube. Put tube on a thermocycler.</p>
| |
| − |
| |
| − | <h5>PCR programme: NebQ5</h5>
| |
| − | <table class="table">
| |
| − | <thead>
| |
| − | <tr>
| |
| − | <th>Step</th>
| |
| − | <th>Temperature in °C</th>
| |
| − | <th>Time in seconds</th>
| |
| − | </tr>
| |
| − | </thead>
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <td>1</td>
| |
| − | <td>98</td>
| |
| − | <td>30</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>2</td>
| |
| − | <td>98</td>
| |
| − | <td>10</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>3</td>
| |
| − | <td>variable - Primer annealing temperature</td>
| |
| − | <td>30</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>4 → 2 30x</td>
| |
| − | <td>72</td>
| |
| − | <td>variable -
| |
| − | 1 min/kb
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>5</td>
| |
| − | <td>72</td>
| |
| − | <td>3 min</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>6</td>
| |
| − | <td>23</td>
| |
| − | <td>10</td>
| |
| − | </tr>
| |
| − |
| |
| − | </tbody>
| |
| − | </table>
| |
| − |
| |
| − | <p>Add 5 µL of 6x Loading Dye for preparative gel electrophoresis.
| |
| − | </p>
| |
| − |
| |
| − |
| |
| − |
| |
| − | <h4 id="goldengate">Golden Gate Assembly</h4>
| |
| − | <p></p>
| |
| − | <h5>Golden Gate Assembly with BsaI</h5>
| |
| − |
| |
| − | <p>For 20µL reaction volume pipette in a PCR Tube:</p>
| |
| − | <table class="table">
| |
| − | <thead>
| |
| − | <tr>
| |
| − | <th>Substance</th>
| |
| − | <th>Amount</th>
| |
| − | </tr>
| |
| − | </thead>
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <td>Empty Backbone 40 nM</td>
| |
| − | <td>1 µL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Backbone Insert(s) 40 nM</td>
| |
| − | <td>2 µL each</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>linear DNA Insert(s) 40 nM</td>
| |
| − | <td>4 µL each</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>GG Mix 20x</td>
| |
| − | <td>1 µL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>GG Buffer 10x</td>
| |
| − | <td>2 µL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>AD</td>
| |
| − | <td>variable</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>total volume</td>
| |
| − | <td>20 µL</td>
| |
| − | </tr>
| |
| − |
| |
| − | </tbody>
| |
| − | </table>
| |
| − |
| |
| − | <p>Vortex and spin down PCR tube. Put tube on a thermocycler.</p>
| |
| − |
| |
| − | <table class="table">
| |
| − | <thead>
| |
| − | <tr>
| |
| − | <th>Step</th>
| |
| − | <th>Temperature in °C</th>
| |
| − | <th>Time in minutes</th>
| |
| − | </tr>
| |
| − | </thead>
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <td>1</td>
| |
| − | <td>37</td>
| |
| − | <td>2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>2 → 1 10x</td>
| |
| − | <td>16</td>
| |
| − | <td>5</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>3</td>
| |
| − | <td>37</td>
| |
| − | <td>10</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>4</td>
| |
| − | <td>55</td>
| |
| − | <td>30</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>5</td>
| |
| − | <td>80</td>
| |
| − | <td>10</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>6</td>
| |
| − | <td>23</td>
| |
| − | <td>10</td>
| |
| − | </tr>
| |
| − |
| |
| − | </tbody>
| |
| − | </table>
| |
| − |
| |
| − | <p>Golden Gate Mix is ready for transformation.
| |
| − | </p>
| |
| − |
| |
| − | <h4 id="goldengate">Golden Gate Assembly</h4>
| |
| − | <p></p>
| |
| − | <h5>Golden Gate Assembly with BsaI / BpiI</h5>
| |
| − |
| |
| − | <p>For 20µL reaction volume pipette in a PCR Tube:</p>
| |
| − | <table class="table">
| |
| − | <thead>
| |
| − | <tr>
| |
| − | <th>Substance</th>
| |
| − | <th>Amount</th>
| |
| − | </tr>
| |
| − | </thead>
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <td>Empty Backbone 40 nM</td>
| |
| − | <td>1 µL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Backbone Insert(s) 40 nM</td>
| |
| − | <td>2 µL each</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>linear DNA Insert(s) 40 nM</td>
| |
| − | <td>4 µL each</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>ATP 10x (aliquoted)</td>
| |
| − | <td>2 µL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>10x CutSmart Buffer</td>
| |
| − | <td>2 µL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>T4 Ligase 10x diluted </td>
| |
| − | <td>1 µL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>BpiI/BsaI</td>
| |
| − | <td>1 µL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>AD</td>
| |
| − | <td>variable</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>total volume</td>
| |
| − | <td>20 µL</td>
| |
| − | </tr>
| |
| − |
| |
| − | </tbody>
| |
| − | </table>
| |
| − |
| |
| − | <p>T4 Ligase has to be diluted 1:10 (0.5 µL T4 and 4.5 µL AD).
| |
| − |
| |
| − | Vortex and spin down PCR tube. Put tube on a thermocycler.
| |
| − | </p>
| |
| − |
| |
| − | <table class="table">
| |
| − | <thead>
| |
| − | <tr>
| |
| − | <th>Step</th>
| |
| − | <th>Temperature in °C</th>
| |
| − | <th>Time in minutes</th>
| |
| − | </tr>
| |
| − | </thead>
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <td>1</td>
| |
| − | <td>37</td>
| |
| − | <td>2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>2 → 1 10x</td>
| |
| − | <td>16</td>
| |
| − | <td>5</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>3</td>
| |
| − | <td>37</td>
| |
| − | <td>10</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>4</td>
| |
| − | <td>55</td>
| |
| − | <td>30</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>5</td>
| |
| − | <td>80</td>
| |
| − | <td>10</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>6</td>
| |
| − | <td>23</td>
| |
| − | <td>10 seconds</td>
| |
| − | </tr>
| |
| − |
| |
| − | </tbody>
| |
| − | </table>
| |
| − |
| |
| − | <p>Golden Gate Mix is ready for transformation.
| |
| − | </p>
| |
| − |
| |
| − |
| |
| − | <h4 id="cpcr">OneTaq Colony PCR</h4>
| |
| − | <p></p>
| |
| − |
| |
| − |
| |
| − | <table class="table">
| |
| − | <thead>
| |
| − | <tr>
| |
| − | <th>Substance</th>
| |
| − | <th>Amount</th>
| |
| − | </tr>
| |
| − | </thead>
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <td>in PCR tube aliquoted oneTaq 2x MM</td>
| |
| − | <td>6 µL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>AD</td>
| |
| − | <td>5 µL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Primer forward</td>
| |
| − | <td>0.5 µL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Primer reverse </td>
| |
| − | <td>0.5 µL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Colony</td>
| |
| − | <td>1 tip</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Total Volume</td>
| |
| − | <td>12 µL</td>
| |
| − | </tr>
| |
| − |
| |
| − | </tbody>
| |
| − | </table>
| |
| − |
| |
| − | <p>Vortex and spin down PCR tube. Put tube on a thermocycler.
| |
| − |
| |
| − | PCR programme: OneTaq
| |
| − | </p>
| |
| − |
| |
| − | <table class="table">
| |
| − | <thead>
| |
| − | <tr>
| |
| − | <th>Step</th>
| |
| − | <th>Temperature in °C</th>
| |
| − | <th>Time in seconds</th>
| |
| − | </tr>
| |
| − | </thead>
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <td>1</td>
| |
| − | <td>94</td>
| |
| − | <td>3 min</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>2</td>
| |
| − | <td>94</td>
| |
| − | <td>30</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>3</td>
| |
| − | <td>variable - Primer annealing temperature</td>
| |
| − | <td>40</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>4 → 2 30x</td>
| |
| − | <td>68</td>
| |
| − | <td>variable - 30 sec/kb</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>5</td>
| |
| − | <td>68</td>
| |
| − | <td>5 min</td>
| |
| − | </tr>
| |
| − |
| |
| − |
| |
| − | </tbody>
| |
| − | </table>
| |
| − |
| |
| − | <p>PCR Mix is ready for electrophoresis. No loading dye is needed.
| |
| − | </p>
| |
| − |
| |
| − |
| |
| − | </div>
| |
| − |
| |
| − | </section>
| |
| | | | |
| | | | |
