Difference between revisions of "Team:EPF Lausanne/Notebook/Protocols"
| Line 149: | Line 149: | ||
<li class="active"><a href="#agarosegel">Agarose Gel</a></li> | <li class="active"><a href="#agarosegel">Agarose Gel</a></li> | ||
<li><a href="#aminoacidsolution">Amino Acid Solution</a></li> | <li><a href="#aminoacidsolution">Amino Acid Solution</a></li> | ||
| − | <li><a href="# | + | <li><a href="#colonypcr">Colony PCR</a></li> |
<li class="dropdown"> | <li class="dropdown"> | ||
<a class="dropdown-toggle" data-toggle="dropdown" href="#">Section 4 <span class="caret"></span></a> | <a class="dropdown-toggle" data-toggle="dropdown" href="#">Section 4 <span class="caret"></span></a> | ||
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</table> | </table> | ||
<p> • Filter and sterilize solutions</p> | <p> • Filter and sterilize solutions</p> | ||
| − | <p> | + | <p> • Add 8 mL per liter of selective medium or spread 500 μL on a selective plate</p> |
</div> | </div> | ||
| − | <!-- | + | <!-- COLONY PCR --> |
| − | <div id=" | + | <div id="colonypcr" class="well"> |
| − | <h1> | + | <h1>Colony PCR (Based on NEB Taq PCR Protocol)</h1> |
| − | + | <h2>Materials</h2> | |
| + | <p> • 10X Standard Taq Reaction Buffer</p> | ||
| + | <p> • dNTPs</p> | ||
| + | <p> • Taq polymerase</p> | ||
| + | <p> • Forward and Reverse Primers</p> | ||
| + | <p> • Nuclease Free Water</p> | ||
| + | <p> • Petri dish with transformed colonies</p> | ||
| + | <h2>Procedure</h2> | ||
| + | <p> • Prepare following reaction in 0.5 mL PCR tubes on ice by making a Master Mix for all reactions and adding polymerase last:</p> | ||
| + | <table width="100%"> | ||
| + | <tr> | ||
| + | <th>Component</th> | ||
| + | <th>25 μL reaction</th> | ||
| + | <th>Final concentration</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>10X Standard Taq Reaction Buffer</td> | ||
| + | <td>2.5 μL</td> | ||
| + | <td>1X</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>10 mM dNTPs</td> | ||
| + | <td>0.5 μL</td> | ||
| + | <td>200 μM</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>10 mM Forward Primer</td> | ||
| + | <td>0.5 μL</td> | ||
| + | <td>0.2 μM</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>10 mM Reverse Primer</td> | ||
| + | <td>0.5 μL</td> | ||
| + | <td>0.2 μM</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Taq DNA Polymerase</td> | ||
| + | <td>0.125 μL</td> | ||
| + | <td>0.625 units/25 μL PCR</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Nuclease Free Water</td> | ||
| + | <td>to 25 μL</td> | ||
| + | <td> </td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | <p> • With a sterile tip, under the flame, scrape part of a single colony and add to PCR tube</p> | ||
| + | <p> • Mix by pipetting up and down or flicking the reactions | ||
| + | <p> • Put tubes in thermocycler with following cycling conditions: | ||
| + | <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 - 60seconds</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> | ||
</div> | </div> | ||
<!-- SECTION 4 --> | <!-- SECTION 4 --> | ||
Revision as of 14:21, 21 July 2015
Protocols
Agarose Gel
Materials
• 1X TAE
• Agarose
• Gel Red
• DNA samples
• 6X loading dye
• Nuclease free water
Procedure
Prepare 1.2% agarose gel for small fragments and 3% agarose gel for large fragments
• Mix 50 mL 1X TAE and 0.6 g (1.2%) or 1.5 g (3%) agarose
• Melt in microwave until agarose has melted (about 50 seconds)
• Add 1.3 μL (1.2%) or 1.5 μL (3%) Gel Red
• Pour solution into agarose gel mold with comb
• Let set for 20 minutes or until solid
• Place gel in 1X TAE and remove comb
• Load samples of 200 ng (or 2 μL) DNA mixed with 2 μL 6X loading dye and nuclease free water up to 12 μL
• Run gel at 100-120 Volts for 40-50 minutes (1.2%) or 80 Volts for 2 hours (3%)
• Take a picture of the gel at the UV detector
Amino acid solution
Materials
• Histidine-Hcl
• Uracil
• Leucine
• Tryptophan
Procedure
| Stock concentration | Final concentration | Total quantity for 50 mL |
|---|---|---|
| 100 mM Histidine-Hcl (209 g/mol) | 20.9 g/L | 0.418 g |
| 20 mM Uracil (112 g/mol) | 2.24 g/L | 0.0448 g |
| 100 mM Leucine (131 g/mol) | 13.1 g/L | 0.262 g |
| 40 mM Tryptophan (204 g/mol) | 8.16 g/L | 0.1632 g |
• Filter and sterilize solutions
• Add 8 mL per liter of selective medium or spread 500 μL on a selective plate
Colony PCR (Based on NEB Taq PCR Protocol)
Materials
• 10X Standard Taq Reaction Buffer
• dNTPs
• Taq polymerase
• Forward and Reverse Primers
• Nuclease Free Water
• Petri dish with transformed colonies
Procedure
• Prepare following reaction in 0.5 mL PCR tubes on ice by making a Master Mix for all reactions and adding polymerase last:
| Component | 25 μL reaction | Final concentration |
|---|---|---|
| 10X Standard Taq Reaction Buffer | 2.5 μL | 1X |
| 10 mM dNTPs | 0.5 μL | 200 μM |
| 10 mM Forward Primer | 0.5 μL | 0.2 μM |
| 10 mM Reverse Primer | 0.5 μL | 0.2 μM |
| Taq DNA Polymerase | 0.125 μL | 0.625 units/25 μL PCR |
| Nuclease Free Water | to 25 μL |
• With a sterile tip, under the flame, scrape part of a single colony and add to PCR tube
• Mix by pipetting up and down or flicking the reactions
• Put tubes in thermocycler with following cycling conditions:
| Step | Temperature | Time | |
|---|---|---|---|
| Initial Denaturation | 95°C | 30 seconds | |
| 25 – 35 cycles | Denaturation | 95°C | 15 – 30 seconds |
| Annealing | 45 – 68°C | 15 - 60seconds | |
| Extension | 68°C | 1 minutes per kb | |
| Final Extension | 68°C | 5 minutes | |
| Hold | 4°C |
Section 4-1
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Section 4-2
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