Team:SDU-Denmark/protocols

From 2010.igem.org

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(Preparation of SOB and SOC media)
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[[Image:Team-SDU-Denmark-PCR_protocol.JPG]]
[[Image:Team-SDU-Denmark-PCR_protocol.JPG]]
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=== CP1.2 ===
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<br>
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Updated Taq protocol for length determination. Due to Taq#s lack of proofreading, only use this protocol for length determination.
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<br>
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''Protocol:''
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<br><br>
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1. A single colony is transfered to each eppendorf tube with a pipette tip. (The same tip is used to plate out on a LA+antibiotic plate afterwards) <br>
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2. Add 30 ul H2O to each tube.<br>
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3. Microwave with open lid at full power for 2 minutes.<br>
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4. Prepare Pre-Mix (number of colonies+1) Distribute 17ul to each tube.<br>
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5. Load and set PCR machine<br>
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6. Ad TAQ polymerase at last moment. Make sure to get it under the surface of the solution.<br>
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7. Run PCR reaction.<br><br>
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''Pre-mix:''
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<br>
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5ul 10x TAQ buffer<br>
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2ul MgCl2 (Increase in 0.25ul incriments if the DNA you want to extract is longer than 3kb.)<br>
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2ul 10pmol/ul forward primer<br>
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2ul 10pmol/ul reverse primer<br>
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1ul 10mM dNTP mix<br>
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7ul H2O<br><br>
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1ul TAQ polymerase -> NB! Pre-Mix is made without TAQ polymerase!<br><br>
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''PCR program''
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<br>
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<table style="text-align: left; width: 394px; height: 200px;" border="1"
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cellpadding="2" cellspacing="2">
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<tbody>
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<tr>
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<td style="vertical-align: top;">1<br>
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</td>
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<td style="vertical-align: top;">Start<br>
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</td>
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<td style="vertical-align: top;">94°C<br>
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</td>
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<td style="vertical-align: top;">2min<br>
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</td>
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</tr>
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<tr>
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<td style="vertical-align: top;">2<br>
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</td>
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<td style="vertical-align: top;">Denaturing<br>
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</td>
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<td style="vertical-align: top;">94°C</td>
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<td style="vertical-align: top;">1min<br>
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</td>
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</tr>
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<tr>
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<td style="vertical-align: top;">3<br>
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</td>
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<td style="vertical-align: top;">Annealing<br>
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</td>
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<td style="vertical-align: top;">55°C<br>
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</td>
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<td style="vertical-align: top;">1min<br>
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</td>
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</tr>
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<tr>
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<td style="vertical-align: top;">4<br>
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</td>
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<td style="vertical-align: top;">Elongation<br>
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</td>
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<td style="vertical-align: top;">72°C<br>
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</td>
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<td style="vertical-align: top;">2min<br>
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</td>
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</tr>
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<tr>
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<td style="vertical-align: top;">5<br>
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</td>
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<td style="vertical-align: top;">GOTO 2<br>
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</td>
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<td style="vertical-align: top;"><br>
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</td>
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<td style="vertical-align: top;">rep. 29x<br>
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</td>
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</tr>
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<tr>
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<td style="vertical-align: top;">6<br>
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</td>
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<td style="vertical-align: top;">End<br>
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</td>
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<td style="vertical-align: top;">72°C<br>
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</td>
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<td style="vertical-align: top;">3min<br>
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</td>
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</tr>
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<tr>
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<td style="vertical-align: top;">7<br>
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</td>
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<td style="vertical-align: top;">Hold<br>
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</td>
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<td style="vertical-align: top;">4°C<br>
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</td>
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<td style="vertical-align: top;">-<br>
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</td>
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</tr>
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</tbody>
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</table>
== Making competent cells of E. coli for transformation ==
== Making competent cells of E. coli for transformation ==

Revision as of 12:57, 13 July 2010