T/A PCR cloning

Steven Sullivan sullivan at gwis2.circ.gwu.edu
Wed Dec 18 18:14:58 EST 1996


severson at uoneuro.uoregon.edu wrote:
: 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.


How long does such a treated plasmid remain stable if frozen?



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