A-tailing of PCR products

[lou]

In article <5u28gd$1f54 at r02n01.cac.psu.edu>, cfb7 at psu.edu wrote:

> Hi,
> I am using a degenerate PCR strategy to amplify fragments of expressed
> members of a subfamily of genes; I am ligating the products into a
> T-vector.  I use a 15' 72C incubation at the end of the PCR cycles to
> add the A-tails.  Is this sufficient to add A-tails to my products or
> should I purify the products and A-tail them in a subsequent reaction
> in which dATP is the only nucleotide available?  Thanks, C-
> 
> Christie Blackman          cfb7 at psu.edu

AS long as your PCR reaction does not contain a proofreading enzyme such as Pwo, Taq will add the necessary A to the 3' end of each strand during the last 10-15 min 72C extension.  We routinely T-A clone in this fashion.

Lou Alarcon
University of Pittsburgh
Dept of Surgery