PCR screening of bacterial colonies
Victor M. Morales
vmorales at BUSTOFF.BWH.HARVARD.EDU
Fri Jun 13 12:59:59 EST 1997
I use a slightly simpler system than those described in previous
answers. I pick a small amount of bacteria with a pipet tip (P200 size)
and shake it into 12.5 microliters of 1xPCR buffer. After overlaying
this suspension with oil, I heat it to 95 degrees for 5 minutes. At
this point I add 12.5 microliters of a solution that is 1xPCR buffer and
2x for the rest of the reaction components (Mg++, dNTPs, primers and Taq
polymerase), mix and continue with the PCR program, usually for 30
cycles. The results seldom have the ghost bands commonly seen after
amplification from whole bacteria.
Brigham & Women's Hospital, Boston, MA. USA.
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