Getting rid of Taq polymerase, HELP
horton at biosci.cbs.umn.edu
Thu Sep 28 11:06:19 EST 1995
jim hartley (jhartley at access2.digex.net) wrote:
: I heard a talk recently on this topic. In an experiment set up to answer
: this question, phenol extraction worked
: very well. Interestingly simply precipitating the PCR product with
: isopropanol and ammonium acetate worked pretty well too. Qiagen and
: glass purification weren't so good. Gel purification before restriction
: wasn't very good either (electroelution was used to get the fragment out
: of the gel slice), so apparently Taq sticks to PCR products. The experiment
: generated a PCR
: product that was part of a drug resistance gene, which was then cut and
: cloned into a plasmid which would theoretically regenerate the drug
: resistance. Filling in the end by residual Taq activity would change the
: reading frame and / or make the fragment unclonable.
Bennett, B.L. and Molenaar, A.J. Cloning of PCR products can be inhibited
by Taq polymerase carryover. BioTechniques 16:32-33, 1994.
(I've got an *autographed* reprint of this one!)
This paper describes experiments to test the hypothesis that Taq
polymerase is carried over into the restriction digestion, fills
in sticky ends, and prevents subsequent ligation. Alcohol precipitation
alone was not sufficient to prevent filling in of 5' overhangs.
Their substrate was cut with EcoRI and BamHI, and fill-in was detected
by incorporation of radionucleotides into the product. Filled-in ends
would not be expected to ligate. Phenol extraction prevented this fill-in.
I have not seen a similar experiment done with a 3' overhang (where
fill-in should not be a problem), though I've heard (or seen) it discussed
somewhere... If anybody has a reference, I'd like to see it.
I'll cross-post this on bionet.journals.letters.biotechniques; its so
*quiet* over there. :)
Robert M. Horton (PhD!) /\ "Crash programs fail because of the theory that
U of M Dermatology Dept || with nine women pregnant you get a baby a month"
Box 98 UMHC, 4-154 PWB /||\ -W. von Braun. Disclaimer: "Bob who?"
Minneapolis, MN 55455 ^^ horton at biosci.cbs.umn.edu (612)625-8941
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