umhosfi1 at cc.umanitoba.ca
Wed Mar 20 23:22:37 EST 1996
In <4icb2f$5oh at taurus.fccc.edu> "Warren D. Kruger" <wd_kruger at fccc.edu> writes:
>We have been having a severe PCR contaimination problem of late. We are
>amplifying a small (134bp) fragment from human DNA and our negative
>control keeps coming up positive. We have thrown away all our old
>reagents, ordered new primers and it is still there. The contamination
>in the water control is usually less intense than the band generated
>from genomic template, but it still interferes with our subsequent
>My question is: Is it possible that the contaiminating DNA is aquired in
>the reaction because our PCR machine itsef is contaminated? Perhaps
>during the heating a little of the contaiminating DNA seeps into the
>stip tubes? Has anyone out there had this happen to them?
>Please reply ASAP. I am desperate to solve this problem.
I have encountered a similar problem with my PCR reactions in the
past and I also through out my primers, buffers, dNTPs and MgCl2 and the
problem persisted. I noticed, however, that the problem was minimized if
I used hot starts and upped my template concentration. In addition, it
is desireable to minimize the number of PCR cycles. I don't know exactly
why this helped, but it did so I was happy. Any suggestions??
More information about the Methods