Colony PCR

Quick and dirty check for correct cloned product!

Make a bacterial cell suspension
Resuspend bacterial colony in 50uL H2O in labeled 1.5mL eppendorf. Just scrape it off with a pipette and pipette up in down in the water.

PCR
Platinum taq (20uL rxn) 0.4uL taq (2 Units/rxn) 2uL 10X buffer 0.8uL 50mM MgCl (25X since want it at 2.5mM) 0.1uL 25mM DNTPs (use at 125uM, so 200X) 1.5uL primer mix (10uM each) 14.1uL H2O 1uL cell suspension

Homemade taq (20uL)
0.2uL taq (100X) 4uL 5X buffer 0.1uL DNTPs 1.5uL primer mix (10uM) 13.2 uL H2O 1uL cell suspension

PCR protocol
95 C for 6 minutes (disrupt cells, separate DNA) Cycle 35 times: 95 C for 30 s (melting) 55 C (or whatever temperature is appropriate) for 30 s (annealing) 72 C for 1 min 45s (elongation) 72 C for 10 minutes (final elongation) 4 C forever