PCR for 6-7 kb
ashok at biochemistry.cwru.edu
Sat Mar 26 00:03:58 EST 1994
In article <2muate$827 at news.univ-rennes1.fr> schweige at sbr (Nina.Schweigert) writes:
>I want to try to make a PCR with DNA fragments wich will result in a
>product of about 6-7 kb.
>I wonder if anyone has tried this before, I know that it should be
>possible, but I don't find any informations about it.
>Who can help me with his own experience or literature?
Take a look at Wally Barnes' paper in the March 15, 1994
issue of PNAS (USA). Unfortunately, I don't remember the
page numbers off-hand. He describes an interesting method
to PCR fragments of upto 35 Kb, using a combination of
an exo(-) and exo(+) thermostable polymerases. He uses
KlenTaq1 and Pfu.
Other modifications were to increase and stabilize the pH
of the reaction buffer, and also decrease the time for
denaturation down to 2 to 20 seconds, probably with the
intention of extending the half-life of the KlenTaq1
There are a number of PCRs in that paper ranging in size
from 6 to 35 kb. A very impressive collection of results.
Ashok Aiyar Mail: ashok at biochemistry.cwru.edu
Department of Biochemistry Tel: (216) 368-3300
CWRU School of Medicine, Cleveland, Ohio Fax: (216) 368-4544
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