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

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

EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits

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-<div class="page-h promotion">
-<div class="container">
-<div class="row">
-<div class="col-md-12 text-center">
-<h3>Protocols</h3>
-</div>
-</div>
-</div>
-</div>
-<body data-spy="scroll" data-target="#myScrollspy" data-offset="20">
-<div class="first-section">
-<div class="row">
-<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="#competentcells">Competent Cell Preparation</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>
-<li><a href="#sdmedium">SD Medium</a></li>
-<li><a href="#transformation">Transformation</a></li>
-<!-- ADD SECTIONS HERE -->
-</ul>
-</nav>
-<div class="col-sm-9">
-<!-- AGAROSE GEL -->
-<div id="agarosegel" class="well">
-<h1>Agarose Gel</h1>
-<h2>Materials</h2>
-<p>• Agarose <input type="checkbox" id="checkbox-1-1" class="regular-checkbox" /><label for="checkbox-1-1"></label></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>
-<!-- COMPETENT CELL PREPARATION -->
-<div id="competentcells" class="well">
-<h1>Competent Cell Preparation – based on Open Wet Ware Protocol</h1>
-<h2>Materials</h2>
-  <p> • Bacterial overnight liquid culture</p>
-  <p> • Lysogeny broth (LB)</p>
-  <p> • CaCl2 solution, ice cold:  60 mM CaCl2, 15% glycerol, 10 mM PIPES, pH 7, filter sterilize and store at room temperature</p>
-<h2>Procedure</h2>
-  <p> • Subculture overnight culture 1:100 in LB</p>
-  <p> • Incubate at 37°C with shaking until culture reaches an OD600 of 0.375</p>
-  <p> • Aliquot 20 mL if the culture into chilled 50 mL tubes</p>
-  <p> • Leave tubes on ice for 5 – 10 minutes</p>
-  <p> • Centrifuge cells at 1600 g for 7 minutes at 4°C</p>
-  <p> • Discard supernatant and resuspend pellet in 4 mL ice cold CaCl2 solution</p>
-  <p> • Centrifuge cells at 1100 g for 5 minutes at 4°C</p>
-  <p> • Discard supernatant and resuspend pellet in 4 mL ice cold CaCl2 solution</p>
-  <p> • Keep on ice for 30 minutes</p>
-  <p> • Centrifuge cells at 1100 g for 5 minutes at 4°C</p>
-  <p> • Discard supernatant and resuspend pellet in 800 μL ice cold CaCl2 solution</p>
-  <p> • Aliquot 100 μL of this suspension into microcentrifuge tubes</p>
-  <p> • Freeze in liquid nitrogen and store at -80°C</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>
-<!-- SD MEDIUM -->
-<div id="sdmedium" class="well">
-<h1>Sd Medium<7h1>
-<h2>Materials</h2>
-  <p> • Amino Acid Powder</p>
-  <p> • Yeast Nitrogen Base</p>
-  <p> • Ammonium Sulphate</p>
-  <p> • Adenine Sulphate</p>
-  <p> • Water</p>
-  <p> • NaOH</p>
-  <p> • Agar</p>
-  <p> • Glucose</p>
-<h2>Procedure</h2>
-  <p> • Place stirrer bar in 2 L Erlenmeyer</p>
-  <p> • Add 2.6 g amino acid powder, 3.4 g yeast nitrogen base, 10 g ammonium sulphate, 1 g adenine sulphate and 950 mL water</p>
-  <p> • Adjust pH to 5.9 by adding a few drops of 10 M NaOH</p>
-  <p> • In an other Erlenmeyer, add 35 g agar and 900 mL water</p>
-  <p> • Autoclave both bottles</p>
-  <p> • Transfer the content of first bottle to the agar-containing bottle</p>
-  <p> • Cool to 55°C</p>
-  <p> • Add 100 ml 40% glucose and 16 ml of the required amino acids</p>
-  <p> • Pour plates</p>
-</div>
-<!-- TRANSFORMATION -->
-<div id="transformation" class="well">
-<h1>Transformation – based on NEB Transformation Protocol</h1>
-<h2>Materials</h2>
-  <p> • Competent cells</p>
-  <p> • DNA</p>
-  <p> • SOC medium (SOB + Glucose)</p>
-  <p> • Petri dish with appropriate antibiotic resistance</p>
-<h2>Procedure</h2>
-  <p> • Thaw competent cells on ice</p>
-  <p> • Add 2 μL DNA to the competent cells, mix by pipetting up and down or flicking the tube 4 -5 times</p>
-  <p> • Place mixture on ice for 30 minutes</p>
-  <p> • Heat shock at 42°C for 30 seconds</p>
-  <p> • Transfer tubes to ice for 2 minutes</p>
-  <p> • Add 950 μL room-temperature SOC media</p>
-  <p> • Incubate at 37°C for 60 minutes with shaking</p>
-  <p> • Spread 100 μL cells onto selection plates (warm plates to 37°C prior to this step for increased efficiency)</p>
-  <p> • Incubate overnight at 37°C</p>
-</div>
-</div>
-</div>
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