Materials
Equipment
Chemicals & consumables
- - Sterile Eppendorf Tubes
- - LB-agar plate with appropriate antibiotic
- - Primers (usually VF2 and VR)
- - Sterile pipet tips
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 positiv control).
Mixtures
1X Reaction Mixture
- - 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)
- - 0,3 µL of Taq DNA Polymerase
- - 0,3 µL of Taq DNA Polymerase
- - 0,3 µL of Taq DNA Polymerase
- - 0,3 µL of Taq DNA Polymerase
PCR programm:
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