Team:TU Darmstadt/protocols/Colony PCR

Short explanation
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
1. Pick one colony with a sterile tip and suspend in 10 µL of ddH2O
2. Inoculate tip with colony into tube. Pipet up and down to ensure all cells are transferred to tube.
3. Start the PCR using the following programm and 1X mix
4. Run a gel to determine the product length (don't forget the positiv control)

Reaction Mix
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
ddH2O to 20 µL
PCR programm: