PCR from single colonies?

Christian Fritze cfritze at cts.com
Wed Jan 17 11:33:58 EST 1996

> > Does anyone have a good protocol or advice for doing PCR directly from
> > single bacterial colonies?

I simply pick up about a glop of cells with a sterile pipette tip (about
0.5 - 1 ul in approximate volume) and scrape it into the bottom of a PCR
tube. When I've  done this for all the colonies I'm interested in, I pipet
pre-mixed PCR cocktail on top (in this case 25 ul for a run in a PE 9600)
and start the PCR run (no mixing required). 25 cycles is plenty. >95%
success rate in my hands. Have found that too many cells inhibits the

I'm using about 50 pM primer, 100 uM dNTPs, standard GIBCO buffer, MgCl2
and Taq.

Christian Fritze
cfritze at cts.com

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