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Team:TU Darmstadt/Notebook/Methods/Colony PCR with Taq polymerase
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| - | <!--TYPO3SEARCH_begin--><div id="c97" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">Colony PCR with Taq | + | <!--TYPO3SEARCH_begin--><div id="c97" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">Colony PCR with Taq Polymerase</h1></div><div><p><b>Equipment:</b></p></div><div><p>- PCR machine</p></div><div></div><div><p><b>Chemicals & consumables:</b></p></div><div><p>- Sterile Eppendorf Tubes</p></div><div><p>- LB-agar plate with appropriate antibiotic</p></div><div><p>- Primers (usually VF2 and VR)</p></div><div><p>- Sterile pipet tips</p><div style="border: 0px; margin: 0px; padding: 0px; font-size: 11px; color: rgb(102, 102, 102); font-family: Tahoma, sans-serif; line-height: 16.5px; "><p style="border: 0px; margin: 0px 0px 10px; padding: 0px; font-size: 13px; text-align: justify; "><b style="border: 0px; margin: 0px; padding: 0px; ">Mixtures: </b>1x Reaction Mixture (25 µL)</p></div><div style="border: 0px; margin: 0px; padding: 0px; font-size: 11px; color: rgb(102, 102, 102); font-family: Tahoma, sans-serif; line-height: 16.5px; "><p style="border: 0px; margin: 0px 0px 10px; padding: 0px; font-size: 13px; text-align: justify; ">- 12,5 µL 2x Taq MM</p></div><div style="border: 0px; margin: 0px; padding: 0px; font-size: 11px; color: rgb(102, 102, 102); font-family: Tahoma, sans-serif; line-height: 16.5px; "><p style="border: 0px; margin: 0px 0px 10px; padding: 0px; font-size: 13px; text-align: justify; ">- 0,5 µL VF2 (10 µM)</p></div><div style="border: 0px; margin: 0px; padding: 0px; font-size: 11px; color: rgb(102, 102, 102); font-family: Tahoma, sans-serif; line-height: 16.5px; "><p style="border: 0px; margin: 0px 0px 10px; padding: 0px; font-size: 13px; text-align: justify; ">- 0,5 µL VR (10 µM)</p></div><div style="border: 0px; margin: 0px; padding: 0px; font-size: 11px; color: rgb(102, 102, 102); font-family: Tahoma, sans-serif; line-height: 16.5px; "><p style="border: 0px; margin: 0px 0px 10px; padding: 0px; font-size: 13px; text-align: justify; ">- 1 µL of colony suspension</p></div><div style="border: 0px; margin: 0px; padding: 0px; font-size: 11px; color: rgb(102, 102, 102); font-family: Tahoma, sans-serif; line-height: 16.5px; "><p style="border: 0px; margin: 0px 0px 10px; padding: 0px; font-size: 13px; text-align: justify; ">- ddH<span style="border: 0px; margin: 0px; padding: 0px; font-size: 10px; line-height: 0; position: relative; vertical-align: baseline; bottom: -0.25em; ">2</span>O to 25 µL</p></div></div><div></div><div><p><b>Procedure:</b></p></div><div></div><div><p>The colony PCR is a modified PCR programm employed to verifiy transformation success by amplifiying the insert or the vector construct used for transformation. This is necessary due to the fact, that a transformation with the empty vector may lead to antibiotic resistance.</p></div><div><p>- Pick one colony with a sterile tip and suspend in 10 µL of ddH2O.</p></div><div><p>- Inoculate tip with colony into tube. Pipet up and down to ensure all cells are transferred to tube.</p></div><div><p>Start the PCR using the following programm with 1x mix and run a gel to determine the PCR product length (don't forget the positive control).</p></div></div><div id="c98" class="csc-default"> |
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Latest revision as of 00:47, 18 October 2014
Colony PCR with Taq Polymerase
Equipment:
- PCR machine
Chemicals & consumables:
- Sterile Eppendorf Tubes
- LB-agar plate with appropriate antibiotic
- Primers (usually VF2 and VR)
- Sterile pipet tips
Mixtures: 1x Reaction Mixture (25 µL)
- 12,5 µL 2x Taq MM
- 0,5 µL VF2 (10 µM)
- 0,5 µL VR (10 µM)
- 1 µL of colony suspension
- ddH2O to 25 µL
Procedure:
The colony PCR is a modified PCR programm employed to verifiy transformation success by amplifiying the insert or the vector construct used for transformation. This is necessary due to the fact, that a transformation with the empty vector may lead to antibiotic resistance.
- Pick one colony with a sterile tip and suspend in 10 µL of ddH2O.
- Inoculate tip with colony into tube. Pipet up and down to ensure all cells are transferred to tube.
Start the PCR using the following programm with 1x mix and run a gel to determine the PCR product length (don't forget the positive control).
| # | Temperature | Time | ||
| 1 | 95°C | 00:05:00 | ||
| 2 | 95°C | 00:00:30 | ||
| 3 | 55°C | 00:00:30 | ||
| 4 | 68°C | 1 min/kbp | ||
| 5 | GO TO 2 | REPEAT 30x | ||
| 6 | 68°C | 1.5 min/kbp | ||
| 7 | 4°C | HOLD |
