Chris_Jones@vme.ccc.nottingham.ac.uk (11/02/90)
I've just seen an ad for Invitrogens PCR cloning kit where they
claim that PCR products always have a 3' A added on. Does anyone
know anything about this (reference?). I have always done a klenow step
before cloning but didn't realise that the ends were ragged because extra
bases were added on? Quite a few of my applications would be wrecked by an
extra A so any tips about eliminating them (if they exist) would be great.
Chris Jones
Biochemistry Dept
Nottingham Univ.
U.K.Chris_Jones@vme.ccc.nottingham.ac.uk (11/05/90)
I've Recently noticed an ad for Invitrogens PCR cloning kit
where they say that Taq always adds an extra A to the 3' end
of a fragment being amplified. Does anyone have any info (reference?)
on this i.e. is it True? I cant find any reference to this activity
in the PCR textbooks. I've successfully cloned lots of PCR products
in the past but as I put sites on the ends I've never looked for extra A's.
Apologies if you've read this before but my mail failed last time
Thanks
Chris Jones
Biochem Dept
Nottingham Univ.WALLEN@CSC.FI (11/06/90)
Dear Chris, A reference that may be of interest to you is Perkin Elmer's newsletter (Amplifications, May 1990, Issue 4). There is an article called "DNA generated by PCR (...) has non-template nucleotide additions: Implications for cloning PCR products. They suggest using Klenow to remove the extra nucleotide (which probably is an A). Hope this is of some help to you. Mika Wallen Univ. Tampere Finland wallen@csc.fi (Internet) wallen@finfun (EARN/Bitnet)
WILSON_R@wums.bitnet (12/14/90)
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