PCR screening of bacterial colonies

Victor M. Morales vmorales at BUSTOFF.BWH.HARVARD.EDU
Fri Jun 13 12:59:59 EST 1997


Hi!
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.

Victor Morales 
Brigham & Women's Hospital, Boston, MA. USA.



More information about the Methods mailing list