blunt cloning of PCR fragments

John Dixon jpcd100 at mole.bio.cam.ac.uk
Tue Aug 1 06:06:30 EST 2000


In article <v04011700b5ab52e0c37d@[144.92.64.174]>, Michael L. Sullivan
<mlsulliv at facstaff.wisc.edu> wrote:

> I've been trying to blunt-end clone some PCR fragments without much
> success.  Controls lead me to beleive that there isn't  problem with the
> ligase or ligation condition.  The PCR fragments were generated using
> KlenTaq LA from Sigma.  Since the enzyme mix is supposed to have a 3' to 5'
> proofreading activity, I figured the fragments should be coming out of the
> reaction with blunt ends.  Am I wrong about that?  Should I go ahead and
> polish up the ends in a separate step anyway?  Thanks for your input.
> 

I recently tried cloning four different Expand Hifi (Roche - Taq+Pwo I
think) generated bands into 2 of Invitrogens Topo cloning kits, 1 for
blunts (ordered by accident) and the followup T tailed version. I got
virtually nothing using the blunt cloning vector, and plenty on the T
tailed one. I think Taq wins the battle over whether a terminal A is
added or not, on the majority of termini. FWIW, I also included a
similar fragment that had been Qiaquicked and stored at -20 for a
couple of weeks, it also worked v poorly in the blunt vector so I guess
that the As stay on for a week or two at least.

Cheers
JD

-- 
John Dixon                    Lab 44 (1223) 334131
Wellcome/CRC Institute        Fax 44 (1223) 334089
Cambridge University
United Kingdom CB2 1QR       e-m: jpcd100 at mole.bio.cam.ac.uk






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