michelle at MOLECULE.BIO.UTS.EDU.AU
Mon Feb 9 18:50:34 EST 1998
On 9 Feb 1998, brett wrote:
> >I have been trying to set up a PCR with bacterial DNA .
> >The problem is that my negative(no DNA) controls are showing up positive on a
> >PCR check gel.
> >I have tried changing all reagents and I have tried 6 different Taqs from
> >different manufacturers!! The negative PCRs still come up with a product!
> >Has anyone of you ever had problems with contaminated Taq??
> >If you have what did you do???
> >I am right now convinced that all Taqs are contaminated.
> WOW! Maybe your H2O, primers, or buffer are contaminated, but these seem more
> likely than 6 tubes of Taq from 6 manufacturers. Back to the bench, and better
> tighten your thinking cap.
I have had a similar problem, which I did isolate to contaminating DNA in
the Taq. The primers were for prokaryotic type rDNA, and although they
were specifically designed for plastid, they amplified a lovely band in
the neg control. I cloned and sequenced it, and it was E.coli SSUrDNA.
Identical, absolutely no question.
SInce it was recombinant Taq, it was
clearly remnants left over from the purification, and given the copy
number of this gene, it wouldn't take much to cause a problem.
I solved the problem by placing the tube of mastermix, including Taq and
primers on the UV transilluminator for 5 minutes on high setting, before
dispensing and adding target DNA. No band! This should fix your problem
perhaps even if the Taq is not to blame.
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