TA cloning of a PCR product generate by Vent DNA polymerase

Dr. Peter Gegenheimer PGegen at UKans.edu
Fri Aug 1 19:58:36 EST 1997


On Fri, 1 Aug 1997 23:44:13, "Dr. Yu" <zyu at unixg.ubc.ca> 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.
> 
> Tai Man Louie
> Dept. of Microbiology,
> Univ. of British Columbia

We routinely use Vent(R) DNA polymerase for site-directed 
mutagenesis by  inverse PCR, after which we circularize the product 
DNA. THe Vent product is blunt-ended and readily ligates to itself; 
a smaller fragment should certainly ligate into any blunt-end 
cloning site. 

I strongly recommend using the simplest reasonable protocol, and 
especially NOT paying any attention to the 1001 commercial gimmicks 
available. The TA plasmids are designed to accept DNA with 
single-base overhangs of T and A. It is beyond me why anyone would 
need to take their clean, blunt-ended DNA and ADD T and A to it, 
just to be able to use an expensive proprietary vector. 

P.G.





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