TA cloning of a PCR product generate by Vent DNA polymerase

Bernard Murray, PhD bernard at elsie.nci.nih.gov
Tue Aug 5 22:26:34 EST 1997


In article <33DF71BE.6DA0 at cellfo.slu.se>, bo.pontoppidan at cellfo.slu.se wrote:

> Dr. Yu wrote:
> > 
> > Does anyone has any successful experience in cloning a PCR product
> > generated by Vent DNA polymerase? The product is 2.7 kbp.  I followed
> > the
> > InVitrogen protocol and incubate the Vent PCR product in Taq at 72 degC
> > for 10 minutes, phenol extract and preciptate the DNA at room temp. The
> > precipitated DNA is resuspended in TE and immediate ligate to the TA
> > vector. I tried this several times but I don't get any clones.
> > 
> Try to do a blunt end ligation directly with the vent pcr product.

Yes, it could be that Taq doesn't like the 3' end of the sequence
and so will not extend (I also assume that you had dATP in with
the Taq).  There have been a few threads in the past concerning the
likelihood of Taq extending a particular sequence and a quick look
through those may reveal if this is the case.
               Bernard

Bernard Murray, PhD
bernard at elsie.nci.nih.gov
Department of Cellular and Molecular Pharmacology, UCSF, USA



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