A-tailing of PCR products

Michael Gerdes mg690769 at bcm.tmc.edu
Thu Aug 28 14:24:40 EST 1997


I have found with TA cloning the most important aspect is to setup the
ligation within 24hrs of the PCR being complete.  You didn't mention what
enzyme you are using for PCR. I have had great luck by- NO purification,
just 2ul from the PCR tube.  I only run a 5 min. polish at the end of the
PCR.

Michael





 >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





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