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Team:TU Darmstadt/Notebook/Methods/Colony PCR with Taq polymerase
From 2014.igem.org
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 |
