Pfu-spiked cycle sequencing

Olav Hungnes ohungnes at bioslave.uio.no
Thu Jun 9 04:06:34 EST 1994


Robert Preston (rapr at MED.PITT.EDU) wrote:
: Forwarded message:
: > From: jspaffor at gpu.srv.ualberta.ca (J. David Spafford)
: > [edit] 
: > I use an ABI sequencer with a 9600 Perkin Elmers PCR machine.  Usually I can
: > get up to 400 bp of good sequence and no more.  ABI provides AmpliTaq with
: > their sequencing kit.  Would I get longer sequence if I spike it with Pfu
: > DNA polymerase?
: > J. David Spafford

: You mean, do Wayne Barnesian Long-PCR factors come into play in the 400-700
: bp range?  Interesting possibility.  Well worth an experiment.  Be sure to
: post the results!


: Rob Preston
: rapr at med.pitt.edu
I don't know much about Pfu, but in the good old days of Vent I was told 
that the 5'to 3' proofreading exonuclease activity would eat at all kinds of 
non-base-paired 3' termini, even the primers to some extent. Isn't there 
a chance that the Pfu will start degrading dideoxy-terminated molecules 
from their 3' end and make a mess of the entire reaction?

Olav
--
_______________________________________________________
Olav Hungnes                     ohungnes at embnet.uio.no
National Institute               Phone  (+47)22042200
of Public Health                 FAX    (+47)22353605
Oslo, NORWAY
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