polishing PCR fragments with T4 DNA polymerase

John Rebers jrebers at nmu.edu
Fri Oct 22 10:58:33 EST 1999


I'm trying to clone some PCR fragments into the Sma I site of pUC19. After
the PCR reaction, I used T4 DNA polymerase to polish the ends, before
kinasing and ligating into the Sma I site. The vector had been treated with
calf intestinal alkaline phosphatase before ligating. In two different
clones, I have come up with missing nucleotides in the insert at the
vector/insert junction - in one case GC was missing from the insert, and in
the other, AC was missing. Since these inserts were amplified with
different primers, I suspect that the T4 DNA polymerase was
over-enthusiastic in the polishing reaction and I lost a couple of nucleotides.

Has anyone else encountered this problem with excess trimming with T4 DNA
polymerase? If so, is Pfu polymerase any better?

Two clones is a small sample size - how likely am I to find clones with the
full-length insert if I just sequence some more of the clones made using T4
polishing?

For the TA cloning enthusiasts, the reading frame after insertion into the
Sma I site is critical for the next experiments with these clones, so I
don't using TA will work.

Thanks,


John Rebers
Department of Cellular Biology
University of Georgia
Athens, GA  30602
jrebers at nmu.edu
706-542-0802 (voice)
706-542-4271 (FAX)
706-583-9618 (home)

(On sabbatical leave from Northern Michigan University until May 2000;
please use contact information above to reach me.)



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