problems with taq-polymerase

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Wed Jun 30 11:57:52 EST 1999


In article <377A22CE.7A4DFF1E at biol.rug.nl>, Eduard Post
<e.post at biol.rug.nl> writes
>
>We have great problems with Taq-polymerase from Amersham Pharmacia. We
>are producing a pcr-product in our negative control ( no DNA added). We
>think the Taq-polymerase is contaminated with DNA, maybe from the
>organism it's isolated from    (Thermus aquaticus). We are performing a
>16s-rDNA pcr with universal-bacterial primers.
>
>Are there more people with the same problem? Have you found a solution?
>Are there other polymerases (from other companies perhaps) wich have the
>same problem?

This is a standard problem with 16s rRNA primers. You are PCRing up the
sheared E.coli DNA carried over in the recombinant Taq purification. You
will usually find it in everyone's Taq but to different degrees. PE sell
a special Taq with low DNA contamination. The problem is that Taq is a
DNA binding protein so hangs onto some xsomal DNA. I'm sure if anyone
checked other recombinant DNA modifying enzymes or maybe even RE's, the
same contaminating DNA problem would also be there. If you know what you
are amplifying them see if you can design primers that will not anneal
to the E.coli 16s rRNA. Difficult I know!

Duncan 
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk



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