PCR questions (the overhang)

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Wed Aug 17 09:34:41 EST 1994


Jim Owens writes in response to LEES at SASK.USASK.CA:
>
LEES>1. What exactly does final extension-used be 10 min after last 
LEES>   cycle-work for?

Jim> The purpose is to make sure that all the PCR-generated fragments are
Jim> completely extended.  10 minutes is overkill; PE-Cetus recommends 5
Jim> minutes.

LEES> 2. Why does PCR product have "A" overhang?

Jim> Taq polymerase tends to add a base to each strand beyond the end of the
Jim> template strand.  It is most frequently an A.  But not always.  See Nucl
Jim> Acids Research 22:2423 (1994).  (This reference came from a post by Brian
Jim> Foley in another PCR thread.)

I've seen it somewhere that the overhang base goes on slowly, so that the 
longer last extension may especially help you if you're trying to do AT 
cloning.  Conversly, a real short last extension may help you if you're
trying to do blunt end cloning.  I haven't personally tested this.

Steve Hardies, Dept. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at thorin.uthscsa.edu




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