PCR for 6-7 kb

[Ashok Aiyar]

In article <2muate$827 at news.univ-rennes1.fr> schweige at sbr (Nina.Schweigert) writes:

>Hi!
>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
polymerase.

There are a number of PCRs in that paper ranging in size
from 6 to 35 kb.  A very impressive collection of results.

Later,
Ashok
--
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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