Pick a colony (0.5-1.5 mm of diameter) and transfer to a tube containing 100 µl , vortex. Add the following components to a 0.2 ml tube (on ice):
Colony lysate | 2.0 µl |
10X PCR buffer (Fermentas) | 5.0 µl |
25 mM | 4.0 µl |
10 mM 4 dNTPs | 1.0 µl |
Taq Polymerase (5U/µl – Fermentas) | 0.5 µl |
30 pmol each primer (30 pmol/µl) | 1.0 µl (X2) |
mili-Q | 36.5 µl |
Total volume | 50.0 µl |
Do one cycle of 5 min at 95ºC and proceed with the amplification cycles