PCR contamination-HELP!

David Hosfield 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 
>analysis.

>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.

>Warrne Kruger


	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??

Dave Hosfield
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