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Direct Cloning of Blunt-end PCR Fragments. HELP !

Peter Gegenheimer peterg at rnaworld.bio.ukans.edu
Fri Jul 1 12:01:00 EST 1994


In <2v1026$s69 at mserv1.dl.ac.uk>, mjones at crc.ac.uk (Dr. M.D. Jones) writes:
 . . . .
>I am trying to clone blunt-ended PCR fragments into SmaI cut M13 vectors.
>So far no luck.  Using the same batch of vector, T4DNA ligase and competent
>cells, I can successfully clune AluI digested lambda DNA fragments.
 . . . .
>The PCR fragments can be digested with restriction enzymes.  I blunt-end the
>fragments with T4DNA polymerase (+/- Klenow).
>
>I have not used T-tailed vectors, as I would like to get blunt-end PCR
>fragments to clone into SmaI cut vectors.
>
>Any help, advice, suggestions, moral support, immoral support, URGENTLY
>neede to keep my insanity intact.

PCR products can be cloned conveniently IF you use a polymerase which generates
blunt ends. VENT polymerase will do this; Taq polymerase will not. A Wayne
Barnes-like mixture of Delta-Taq plus VENT might also work. VENT is a finicky 
enzyme compared with Taq, but its 10-folder lower error frequency and its
production of blunt-ended products warrants the effort. Talk to the NEB people
to get latest info on VENT.

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