PCR screening of bacterial colonies
yoonm at fri.cri.nz
Wed Jun 18 22:25:57 EST 1997
In my experience, there has been no need for any other manipulations (such
as boiling) other than just touching the colonies with sterile toothpicks
and then swirling it directly in the PCR reaction mix. I always get clear
results with that.
In article <33A1B4AD.1DEB at bustoff.bwh.harvard.edu>,
75 at bustoff.bwh.harvard.edu, Francis at bustoff.bwh.harvard.edu,
Street at bustoff.bwh.harvard.edu, Boston at bustoff.bwh.harvard.edu,
MA at bustoff.bwh.harvard.edu, 02115 at bustoff.bwh.harvard.edu wrote:
> 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.
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