A-tailing of PCR products

[Ulrich Maier]

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
>Department of Biochemistry and Molecular Biology
>Penn State University
>University Park, PA
>"There are alot of bastards in the world, the number increasing the 
>further one gets from Missoula, Montana."  
>           Norman McLean, A River Runs Through It

IMHO it is not necessary to make a final extension step for adding a
A-tail to your PCR-product. Even without this step ~80% of your
product should be tailed if you are using a polymerase without proof
reading activity.

Yours,

Ulrich Maier