COLONY PCR

E coli colony PCR protocol, adapted from https://www.pombevolution.eu/labwiki/index.php/E_coli_colony_PCR

Use pipette tips instead of toothpicks (picks fewer bacteria), to place bacteria in 50 μl water, in a PCR tube. Boil in the PCR machine for 10 min (use ‘BOIL’ program). Then use 1 μl as a template in the following reaction:

PCR mix/reaction:

H2O                                              11.45 μl

Taq PCR Buffer 10X                        1.5 μl

dNTPs (10 mM each)                      0.3 μl

Primer Fwd (50 μM)                        0.3 μl

Primer Rev (50 μM)                        0.3 μl

Taq polymerase (5U/μl)                0.15 μl

Run the ‘COLONY PCR’ program (it is the same as shown here https://www.pombevolution.eu/labwiki/index.php/E_coli_colony_PCR)