You can expect 500-800 bases without any N's if you follow our methods and
run a 48cm wtr gel on the 377 using any comb up to 64 lanes (the extent of
our experience). From your description of the variable results problem, you
may need to focus more on primer selection.
Hope this helps,
Al
******************
Alfred M. Cairo
BACPAC Resources
DNA Sequencing Lab
Dept. of Cancer Genetics
Roswell Park Cancer Institute
Buffalo NY 14263
email: cairo@dejong.med.buffalo.edu
web site: http://bacpac.med.buffalo.edu
phone: 716-845-4543
-----Original Message-----
From: Leviten, Dina <dleviten@icos.com>
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Date: Thursday, June 10, 1999 8:13 PM
Subject: BAC sequencing
>Hello All!
>
>What protocols are out there that describe: the preparation of the BAC
DNA,
>clean-up of DNA (if necessary), and the PCR reaction set-up? We could use
>any advice!
>
>We have been sequencing directly from the BAC clone and have been getting
>variable results. Either it works or it doesn't. Out of about different
50
>primers, 12 have given good sequence. I just ran 10 primers on the 3700,
>and 1 gave excellent sequence (700bp), 1 gave poor sequence (50bp) and the
>others did not work. (This is about the same success rate as the 373S.)
>Just so you know, we have been using 1-2ug of DNA, ~12pmols of primer and
>adding extra enzyme. We have tried two different PCR protocols and the one
>at least gives us these hit-and-miss results. We are thinking resorting to
>shotgun cloning it and just sequencing the whole thing. Let me know what
>you think...
>
>Thanks in advance!
>Dina Leviten
>ICOS, Corp.
>Bothell, WA
>
>