polishing PCR fragments with T4 DNA polymerase

Francesca Catalano fcatala at luc.edu
Thu Oct 28 19:34:08 EST 1999


By "polish" do you mean eliminate any A's that were generated during the
Taq PCR step?  If so.. try using Pfu polymerase.  It does not add any
extra A's.
Francesca Catalano
fcatala at luc.esu

Bas Jansen wrote:
> 
> John Rebers heeft geschreven in bericht
> <4.1.19991022113710.00988cf0 at pop.mail.nmu.edu>...
> >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.
> 
> Have you used PAGE-purified primers for your PCR? If not, these shorter
> inserts may well be the result of n-1 primers.
> 
> Best,
> Bas



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