TA cloning of a PCR product generate by vent DNA polymerase

CHANDRA KRISHNAN CSK1 at sigsmtp.sial.com
Wed Jul 30 09:35:44 EST 1997



If my memory serves me right, Vent polymerase has both exonuclease
activities.  The proof reading activity does not allow the addition of A's on
the tails of the PCR product, which is desirable for TA cloning vector. 
Switch your enzyme to regular Taq or your vector to a blunt ended one.
Talking to Stratagene about this may be a better idea.

Chandra Krishnan Ph. D.
Sigma-Aldrich
csk1 at sigsmtp.sial.com

>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




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