Difference between revisions of "Team:EPF Lausanne/Test"

 
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                                <li><a href="https://2015.igem.org/Team_EPF_Lausanne/Project/Background">Background</a></li>
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                                <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Project/Description">Description</a></li>
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                                <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Project/Applications">Applications</a></li>
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                                <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Project/Modelling">Modelling</a></li>
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                            <li class="dropdown">
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                            <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-haspopup="true" aria-expanded="false">Achievements <span class="caret"></span></a>
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                                <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Achievements/Vivo">In vivo</a></li>
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                                <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Achievements/Silico">In silico</a></li>
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                                <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Achievements/Judging">Judging</a></li>
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                                <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Achievements/Parts">Parts</a></li>
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                                <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Policy/Ethic">Ethics</a></li>
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                                <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Notebook/Ecoli">E. Coli</a></li>
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                                <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Notebook/Yeast">Yeast</a></li>
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                                <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Notebook/Protocols">Protocols</a></li>
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                                <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Team/Meet">Meet us</a></li>
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                                <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Timeline">Timeline</a></li>
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                                <li><a href="https://2015.igem.org/Team:EPF_Lausanne/Team/Aknowledgements">Aknowledgements</a></li>
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                    </div>
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                </nav>       
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            </div>
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         </div>
 
         </div>
    </div> <!-- .site-header -->
+
        <div class="item">
               
+
          <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2015/4/4b/EPF_Lausanne_Home2.png" alt="Second slide">
           
+
 
+
    <div class="page-h promotion">
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        <div class="container">
+
            <div class="row">
+
                <div class="col-md-12 text-center">
+
                    <h3>Protocols</h3>
+
                </div>
+
            </div>
+
 
         </div>
 
         </div>
    </div>
+
         <div class="item">
 
+
          <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2015/9/9b/EPF_Lausanne_Home3.png" alt="Third slide">
    <body data-spy="scroll" data-target="#myScrollspy" data-offset="20">
+
    <div class="first-section">
+
         <div class="row">
+
            <div data-spy="affix" data-offset-top="60" data-offset-bottom="200">
+
            <nav class="col-sm-3" id="myScrollspy">
+
              <ul class="nav nav-pills nav-stacked">
+
                <li class="active"><a href="#agarosegel">Agarose Gel</a></li>
+
                <li><a href="#aminoacidsolution">Amino Acid Solution</a></li>
+
                <li><a href="#colonypcr">Colony PCR</a></li>
+
                <li><a href="#gibsonassembly">Gibson Assembly</a></li>
+
                <li><a href="#miniprep">Miniprep</a></li>
+
                <li class="dropdown">
+
                  <a class="dropdown-toggle" data-toggle="dropdown" href="#">Polymerase Chain Reaction (PCR)<span class="caret"></span></a>
+
                  <ul class="dropdown-menu">
+
                    <li><a href="#phusionpcr">Phusion PCR</a></li>
+
                    <li><a href="#taqpcr">Taq PCR</a></li>                   
+
                  </ul>
+
                </li>
+
    <li><a href="#pcrpurification">PCR Product Purification</a></li>
+
    <li><a href="#pegliac">PEG/LiAc Solution</a></li>
+
 
+
    <!-- ADD SECTIONS HERE -->
+
       
+
 
+
    </ul>
+
            </nav></div>
+
            <div class="col-sm-9">
+
 
+
    <!-- AGAROSE GEL -->
+
              <div id="agarosegel" class="panel">   
+
                <h1>Agarose Gel</h1>
+
            <h2>Materials</h2>
+
        <p> • 1X TAE</p>
+
        <p> • Agarose</p>
+
        <p> • Gel Red</p>
+
        <p> • DNA samples</p>
+
        <p> • 6X loading dye</p>
+
        <p> • Nuclease free water</p>
+
        <h2>Procedure</h2>
+
        <p>Prepare 1.2% agarose gel for small fragments and 3% agarose gel for large fragments</p>
+
        <p> • Mix 50 mL 1X TAE and 0.6 g (1.2%) or 1.5 g (3%) agarose</p>
+
        <p> • Melt in microwave until agarose has melted (about 50 seconds)</p>
+
        <p> • Add 1.3 μL (1.2%) or 1.5 μL (3%) Gel Red</p>
+
        <p> • Pour solution into agarose gel mold with comb</p>
+
        <p> • Let set for 20 minutes or until solid</p>
+
        <p> • Place gel in 1X TAE and remove comb</p>
+
        <p> • Load samples of 200 ng (or 2  μL) DNA mixed with 2  μL 6X loading dye and nuclease free water up to 12  μL</p>
+
        <p> • Run gel at 100-120 Volts for 40-50 minutes (1.2%) or 80 Volts for 2 hours (3%)</p>
+
        <p> • Take a picture of the gel at the UV detector</p>
+
              </div>
+
 
+
    <!-- AMINO ACID SOLUTION -->
+
              <div id="aminoacidsolution" class="well">
+
                <h1>Amino acid solution</h1>
+
        <h2>Materials</h2>
+
        <p> • Histidine-Hcl</p>
+
        <p> • Uracil</p>
+
        <p> • Leucine</p>
+
        <p> • Tryptophan</p>
+
        <h2>Procedure</h2>
+
        <table width="100%">
+
            <tr>
+
                <th>Stock concentration</th>
+
                <th>Final concentration</th>
+
                <th>Total quantity for 50 mL</th>
+
            </tr>
+
            <tr>
+
                <td>100 mM Histidine-Hcl (209 g/mol)</td>
+
                <td> 20.9 g/L</td>
+
                <td> 0.418 g</td>
+
            </tr>
+
            <tr>
+
                <td>20 mM Uracil (112 g/mol)</td>
+
                <td> 2.24 g/L</td>
+
                <td> 0.0448 g</td>
+
            </tr>
+
            <tr>
+
                <td>100 mM Leucine (131 g/mol)</td>
+
                <td> 13.1 g/L</td>
+
                <td> 0.262 g</td>
+
            </tr>
+
            <tr>
+
                <td>40 mM Tryptophan (204 g/mol)</td>
+
                <td> 8.16 g/L</td>
+
                <td> 0.1632 g</td>
+
            </tr> 
+
        </table>   
+
        <p> • Filter and sterilize solutions</p>
+
        <p> • Add 8 mL per liter of selective medium or spread 500 μL on a selective plate</p>
+
              </div>       
+
 
+
<!-- COLONY PCR -->
+
<div id="colonypcr" class="well">       
+
<h1>Colony PCR</h1>
+
<h2>Materials</h2>
+
    <p> • Materials for Taq PCR (except template plasmid DNA)</p>
+
    <p> • Petri dish with transformed colonies</p>
+
<h2>Procedure</h2>
+
    <p> • Prepare 25 μL reactions as in Taq PCR Protocol without template DNA</p>
+
            <p> • With a sterile tip, under the flame, scrape part of a single colony and add to PCR tubes</p>
+
    <p> • Mix by pipetting up and down or flicking the reactions
+
    <p> • Put tubes in thermocycler with cycling conditions as described in Taq PCR Protocol</p>
+
</div>
+
 
+
    <!-- GIBSON ASSEMBLY -->
+
<div id="gibsonassembly" class="well">
+
<h1>Gibson Assembly - based on NEB Gibson Assembly Protocol</h1>
+
<h2>Materials</h2>
+
    <p> • DNA fragments</p>
+
    <p> • 2X Gibson Assembly Mater Mix (NEB)</p>
+
    <p> • 2X NEBuilder Positive Control (NEB)</p>
+
    <p> • Deionized water</p>
+
<h2>Procedure</h2>
+
    <p> • Set up following reactions on ice, adding Gibson Assembly Master Mix last:</p>
+
    <table width="100%">
+
        <tr>
+
        <th>Component</th>
+
        <th>2 – 3 Fragments Assembly</th>
+
        <th>4 – 6 Fragments Assembly</th>
+
        <th>Positive Control</th>
+
        </tr>
+
        <tr>
+
        <td>Total Amount of Fragments</td>
+
        <td>0.02 – 0.5 pmols</td>
+
        <td>0.2 – 1 pmols</td>
+
        <td>10 μL</td>
+
        </tr>
+
        <tr>
+
        <td>2X Gibson Assembly Master Mix</td>
+
        <td>10 μL</td>
+
        <td>10 μL</td>
+
        <td>10 μL</td>
+
        </tr>
+
        <tr>
+
        <td>Deionized water  </td>
+
        <td>to 20 μL</td>
+
        <td>to 20 μL</td>
+
        <td>0 μL</td>
+
        </tr>
+
    </table>
+
    <p>          Optimized efficiency for 50 – 100 ng of vectors and 2 – 3 fold of excess inserts</p>
+
    <p> • Incubate samples at 50°C for 15 minutes (2 – 3 fragments) or for 60 minutes (4 – 6 fragments)</p>
+
    <p> • Store samples on ice or at -20°C until transformation</p>
+
    <p> • Transform competent cells following the Transformation Protocol</p>
+
</div>
+
 
+
<!-- MINIPREP -->
+
<div id="miniprep" class="well">       
+
<h1>Miniprep - with QIAprep Spin Miniprep Kit (QIAGEN)</h1>
+
<h2>Materials</h2>
+
    <p> • Bacterial overnight liquid cultures (1 - 5 mL)
+
    <p> • QIAprep Spin Miniprep Kit
+
<h2>Procedure</h2>
+
    <p> • Pellet 1 -5 mL bacterial culture by centrifugation at more than 8000 rpm for 3 minutes</p>
+
    <p> • Resuspend pelleted bacterial cells in 250 μL P1 buffer and transfer to a microcentrifuge tube</p>
+
    <p> •  Add 250 μL P2 buffer and mix by inverting tube 4 – 6 times</p>
+
    <p> • Add 350 μL N3 buffer and mix by inverting tube 4- 6 times</p>
+
    <p> • Centrifuge for 10 min at 13000 rpm</p>
+
    <p> • Apply supernatant to the QIAprep spin column by pipetting, centrifuge for 30 – 60 seconds and discard flow-through</p>
+
    <p> • Wash the QIAprep spin column by adding 0.5 mL PB buffer, centrifuge for 30 – 60 seconds and discard flow-through</p>
+
    <p> • Wash the QIAprep spin column by adding 0.75 mL PE buffer, centrifuge for 30 – 60 seconds and discard flow-through</p>
+
    <p> • Centrifuge for 1 minute to remove residual wash buffer</p>
+
    <p> •  Elute DNA by placing QIAprep column in a clean 1.5 mL microcentrifuge tube and adding 50 μL EB buffer or water (or less for higher concentration). Let stand for 1 minute and centrifuge for 1 minute</p>
+
</div>
+
 
+
<!-- PHUSION PCR -->
+
<div id="phusionpcr" class="well">               
+
<h1>Phusion PCR – based on NEB Phusion PCR Protocol</h1>
+
<h2>Materials</h2>
+
    <p> • 5X Phusion HF or GC Buffer</p>
+
    <p> • dNTPs</p>
+
    <p> • Forward and Reverse Primers</p>
+
    <p> • Template plasmid DNA</p>
+
    <p> • Phusion DNA polymerase</p>
+
    <p> • Nuclease Free Water</p>
+
<h2>Procedure</h2>
+
    <p> • Prepare following reaction in 0.5 mL PCR tubes on ice, adding polymerase last:</p>
+
    <table width="100%">
+
    <tr>
+
        <th>Component</th>
+
        <th>20 μL reaction</th>
+
        <th>50  μL reaction</th>
+
    </tr>
+
    <tr>
+
        <td>5X Phusion HF or GC Buffer</td>
+
        <td>4 μL</td>
+
        <td>10 μL</td>
+
    </tr>
+
    <tr>
+
        <td>10 mM dNTPs</td>
+
        <td>0.4 μL</td>
+
        <td>1 μL</td>
+
    </tr>
+
    <tr>
+
        <td>10 mM Forward Primer</td>
+
        <td>1 μL</td>
+
        <td>2.5 μL</td>
+
    </tr>
+
    <tr>
+
        <td>10 mM Reverse Primer</td>
+
        <td>1 μL</td>
+
        <td>2.5 μL</td>
+
    </tr>
+
    <tr>
+
        <td>Template plasmid DNA</td>
+
        <td>1 pg – 10 ng</td>
+
        <td>1 pg – 10 ng</td>
+
    </tr>
+
    <tr>
+
        <td>Phusion DNA Polymerase</td>
+
        <td>0.2 μL</td>
+
        <td>0.5 μL</td>
+
    </tr>
+
    <tr>
+
        <td>Nuclease Free Water</td>
+
        <td>to 20 μL</td>
+
        <td>to 50 μL</td>
+
    </tr> 
+
    </table>
+
            <p>        Usually 100 pg – 1 ng of template DNA is sufficient</p>
+
    <p> • Mix by pipetting up and down or flicking the reactions
+
    <p> • Put tubes in thermocycler (with a pre-heated lid) with following cycling conditions:</p>
+
    <table width="100%">
+
    <tr>
+
        <th>Step</th>
+
        <th>  </th>
+
        <th>Temperature</th>
+
        <th>Time</th>
+
    </tr>
+
    <tr>
+
        <td>Initial Denaturation</td>
+
        <td>  </td>
+
        <td>98°C</td>
+
        <td>30 seconds</td>
+
    </tr>
+
    <tr>
+
        <td>25 – 35 cycles</td>
+
        <td>Denaturation</td>
+
        <td>98°C</td>
+
        <td>5 - 10 seconds</td>
+
    </tr>
+
    <tr>
+
        <td>  </td>
+
        <td>Annealing</td>
+
        <td>45 – 72°C</td>
+
        <td>10 – 30 seconds</td>
+
    </tr>
+
    <tr>
+
        <td>  </td>
+
        <td>Extension</td>
+
        <td>72°C</td>
+
        <td>15 -30 seconds per kb</td>
+
    </tr>
+
    <tr>
+
        <td>Final Extension</td>
+
        <td>  </td>
+
        <td>72°C</td>
+
        <td>5 -10 minutes</td>
+
    </tr>
+
    <tr>
+
        <td>Hold</td>
+
        <td>  </td>
+
        <td>4°C </td>
+
        <td>  </td>
+
    </tr> 
+
    </table>
+
<h2>Guidelines</h2>
+
    <p>To be completed</p>
+
</div>
+
 
+
<!-- TAQ PCR -->
+
<div id="taqpcr" class="well">
+
<h1>Taq PCR – based on NEB Taq PCR Protocol</h1>
+
<h2>Materials</h2>
+
    <p> • 10X Standard Taq Reaction Buffer</p>
+
    <p> • dNTPs</p>
+
    <p> • Forward and Reverse Primers</p>
+
    <p> • Template plasmid DNA</p>
+
    <p> • Taq DNA polymerase</p>
+
    <p> • Nuclease Free Water</p>
+
<h2>Procedure</h2>
+
    <p> • Prepare following reaction in 0.5 mL PCR tubes on ice, adding polymerase last:</p>
+
    <table width="100%">
+
    <tr>
+
        <th>Component</th>
+
        <th>25 μL reaction</th>
+
        <th>50 μL reaction</th>
+
    </tr>
+
    <tr>
+
        <td>10X Standard Taq Reaction Buffer</td>
+
        <td>2.5 μL</td>
+
        <td>5 μL</td>
+
    </tr>
+
    <tr>
+
        <td>10 mM dNTPs</td>
+
        <td>0.5 μL</td>
+
        <td>1 μL</td>
+
    </tr>
+
    <tr>
+
        <td>10 mM Forward Primer</td>
+
        <td>0.5 μL</td>
+
        <td>1 μL</td>
+
    </tr>
+
    <tr>
+
        <td>10 mM Reverse Primer</td>
+
        <td>0.5 μL</td>
+
        <td>1 μL</td>
+
    </tr>
+
    <tr>
+
        <td>Template plasmid DNA</td>
+
        <td>1 pg – 1 ng</td>
+
        <td>1 pg – 1 ng</td>
+
    </tr>
+
    <tr>
+
        <td>Taq DNA Polymerase</td>
+
        <td>0.125 μL</td>
+
        <td>0.25 μL</td>
+
    </tr>
+
    <tr>
+
        <td>Nuclease Free Water</td>
+
        <td>to 25 μL</td>
+
        <td>to 50 μL</td>
+
    </tr> 
+
    </table>
+
            <p>        Usually 100 pg – 1 ng of template DNA is sufficient</p>
+
    <p> • Mix by pipetting up and down or flicking the reactions
+
    <p> • Put tubes in thermocycler (with a pre-heated lid) with following cycling conditions:</p>
+
    <table width="100%">
+
    <tr>
+
        <th>Step</th>
+
        <th>  </th>
+
        <th>Temperature</th>
+
        <th>Time</th>
+
    </tr>
+
    <tr>
+
        <td>Initial Denaturation</td>
+
        <td>  </td>
+
        <td>95°C</td>
+
        <td>30 seconds</td>
+
    </tr>
+
    <tr>
+
        <td>25 – 35 cycles</td>
+
        <td>Denaturation</td>
+
        <td>95°C</td>
+
        <td>15 – 30 seconds</td>
+
    </tr>
+
    <tr>
+
        <td>  </td>
+
        <td>Annealing</td>
+
        <td>45 – 68°C</td>
+
        <td>15 – 60 seconds</td>
+
    </tr>
+
    <tr>
+
        <td>  </td>
+
        <td>Extension</td>
+
        <td>68°C</td>
+
        <td>1 minutes per kb</td>
+
    </tr>
+
    <tr>
+
        <td>Final Extension</td>
+
        <td>  </td>
+
        <td>68°C</td>
+
        <td>5 minutes</td>
+
    </tr>
+
    <tr>
+
        <td>Hold</td>
+
        <td>  </td>
+
        <td>4°C </td>
+
        <td>  </td>
+
    </tr> 
+
    </table>
+
<h2>Guidelines</h2>
+
    <p>To be completed</p>
+
</div>
+
 
+
<!-- PCR PURIFICATION -->
+
<div id="pcrpurification" class="well">       
+
<h1>PCR Product Purification - with QIAquick PCR Purification Kit (QIAGEN)</h1>
+
<h2>Materials</h2>
+
    <p> • PCR products</p>
+
    <p> • QIAquick PCR Purification Kit</p>
+
<h2>Procedure</h2>
+
    <p> • Add 5 volumes PB buffer to 1 volume of PCR product and mix</p>
+
    <p> • Place QIAquick column in 2 ml collection tube</p>
+
    <p> • Apply samples to QIAquick column and centrifuge for 30 – 60 seconds, discard flow-through</p>
+
    <p> • Wash by adding 0.75 μL PE buffer to QIAquick column and centrifuge fo 30 – 60 seconds, discard flow-through</p>
+
    <p> • Centrifuge QIAquick column for 1 minutes to remove residual wash buffer</p>
+
    <p> • Elute DNA by adding 30 or 50 μL EB buffer or water to the center of the QIAquick column. Let stand for 1 minutes and centrifuge for 1 minute</p>
+
</div>
+
 
+
<!-- PEG LIAC SOLUTION -->
+
<div id="pegliac" class="well">       
+
<h1>PEG/LiAc Solution</h1>
+
<h2>Materials</h2>
+
    <p> • 50% PEG  (Polyethylene glycol) prepared with sterile deionized water</p>
+
    <p> • 10X TE buffer: 0.1 M Tris-Hcl, 10 mM EDTA, ph 7.5, autoclaved</p>
+
    <p> • 10X LiAc: 1 M lithium acetate, pH 7.5 adjusted with dilute acetic acid, autoclaved</p>
+
<h2>Procedure</h2>
+
    <p> • Prepare PEG/LiAc solution as follows:</p>
+
    <table width="100%">
+
    <tr>
+
        <th>Stock concentration</th>
+
        <th>Final concentration</th>
+
        <th>Total quantity for 10 mL solution</th>
+
    </tr>
+
    <tr>
+
        <td>50% PEG</td>
+
        <td>40% PEG</td>
+
        <td>8 mL</td>
+
    </tr>
+
    <tr>
+
        <td>10X TE buffer</td>
+
        <td>1X TE buffer</td>
+
        <td>1 mL</td>
+
    </tr>
+
    <tr>
+
        <td>10X LiAc</td>
+
        <td>1X LiAc</td>
+
        <td>1 mL</td>
+
    </tr>
+
    </table>   
+
</div>
+
 
+
 
+
 
+
 
+
 
+
 
+
 
+
   
+
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+
 
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          <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2015/1/1a/EPF_Lausanne_Home4.png" alt="Fourth slide">
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Latest revision as of 11:07, 16 September 2015

EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits

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