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Team:TU Darmstadt/Notebook/Methods/Colony PCR with Taq polymerase
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Revision as of 16:03, 16 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 and 1X mix.
Run a gel to determine the 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 |
