T/A PCR cloning

severson at uoneuro.uoregon.edu severson at uoneuro.uoregon.edu
Thu Nov 28 20:10:19 EST 1996


In article <329D948E.776D at udcf.gla.ac.uk>, gbga14 at udcf.gla.ac.uk wrote:

> Does anyone have a method for adding a single T to the 3'OH of blunt cut 
> plasmid for use in A/T Tac generated PCR fragment cloning.  I am working 
> on the assumption that dideoxy T and terminal transferase are used, 
> however a protocol known to work would be very useful

Taq likes to stick As on the 3' ends of DNA strands, but if only dTTP is
available, Taq will add a T overhang.  After your digestion, set up a
"PCR" reaction with Taq and ONLY dTTP and incubate a couple of hours at
your preferred extension time.



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