Team:TU Darmstadt/Protocols/Colony PCR
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Contents |
Colony PCR
About
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.
Materials
- Sterile Eppendorf Tubes
- LB-agar plate with appropriate antibiotic
- Primers (usually VF2 and VR)
- PCR machine
- Sterile pipet tips
Procedure
- Pick one colony with a sterile tip and suspend in 10 µL of DI H2O
- 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 positiv control)
Reaction mixture
1X reaction mix contains:
- 2 µL of 10x Thermopol Reaction Buffer
- 0,4 µL of dNTPs (10 mM each)
- 0,3 µL of Taq DNA Polymerase
- VF2 (10 pmol)
- VR (10 pmol)
- 0,6 µL of DMSO
- 1 µL of colony suspension
- DI water to 20 µL
- PCR program
# Temperature Time 1 95 °C 00:05:00 2 95 °C 00:00:20 3 62 °C 00:00:30 4 68 °C 00:02:00 5 GO TO 2 REPEAT 30x 6 68 °C 00:05:00 7 4 °C HOLD
Elongation time (step 4 depends on the length of the fragment 1 min per kb)).