PCR cloning

WILSON_R at wums.bitnet WILSON_R at wums.bitnet
Thu Dec 13 15:04:00 EST 1990


Hi!

I thought I might say a word or two in response to recent inquiries
about cloning PCR products.

As far as the concept of Taq DNA pol leaving an overhanging A residue
at the 3' end of PCR products, I'd say that the A (or some other base)
is not there in some percentage of the DNA.  You certainly can clone
PCR products in a blunt end site with very good efficiency with no other
post- (or pre-) PCR treatment than kinasing.

If we add T4 DNA pol to the PCR mixture immediately following PCR, and
incubate at 37 degrees for 15 minutes, cloning efficiency is doubled.
Thus, your overhanging base.  A 1-hour treatment at 15 degrees with a
few units of Klenow seems to work fine also.

You guys not wanting to spend the bucks on those TA cloning kits
might want to consider this method.

Good Luck!

R. Wilson, St. Louis





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