polishing PCR fragments with T4 DNA polymerase

Bas Jansen bas at nospam.nl
Mon Oct 25 13:42:23 EST 1999


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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