PCR Screening of transformants?

V. Blaschke vblasch at gwdg.de
Fri Dec 5 15:13:41 EST 1997


Hi,

I routinely pick colonies into 50 µl of LB-media, vortex vigorously and
take 2 µl into my PCR mix. Cycling protocol is a for normal PCR, i.e. an
initial 2 min. at 95, and then 35 cycles of 45 sec. 95, 45 sec.
annealing temp and 1 min. 30 sec. for extension. Works perfect and has
never failed me. Once I have the PCR results, I then do o/n cultures by
adding the remaining 48 µl to 5 ml LB and prep plasmid the next day. So
far, I never had a false-positive clone by PCR.

Good luck!

  Volker



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