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
reaction.

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

-- 
Christian Fritze
cfritze at cts.com



More information about the Methods mailing list