Kathy,
Whenever I see broad peaks on our 3700 I take 5ul of the remaining sample,
add another 5ul of formamide and re-run. I usually see 700+ bases as long
as we re-run within 48hrs of the initial addition of formamide.
I think it's down to overloading of the capillary - if the problems are the
same. We have to dilute more for constructs above 7kb or, more usually,
drop input template amounts to 40ng/kb. This works for constructs from 7kb
plasmids all the way up to 40kb cosmids in our hands.
Regards,
Stuart.
Stuart Bayliss,
Manager,
Genetics Core Facility,
MRC Clinical Science Centre,
Hammersmith Hospital,
Du Cane Road,
London.
W12 0NN
Tel (lab): 020 8383 3181
Tel (office): 020 8383 8305
Fax: 020 8383 8338
email: stuart.bayliss@csc.mrc.ac.uk
web: http://gcf.csc.mrc.ac.uk
-----Original Message-----
From: kathyi@cgl.ucsf.edu [mailto:kathyi@cgl.ucsf.edu]
Sent: Tuesday, January 09, 2001 1:39 AM
To: Recipients of ABRF List
Cc: sandifer@cgl.ucsf.edu
Subject: DNA sequencing problems
Is anyone having problems or successes sequencing a modified bluescript
containing a yeast expression vector plus insert on an ABI 3700. One user
reports that his templates work on a ABI 310, but that the same sample give
very broad peakson our ABI 3700, indicative of some degree of capillary
clogging on our 3700. Both runs are with Big Dye chemistry.
Thanks for your input/suggestions.
Kathy
Kathryn Ivanetich, Ph.D.
Acting Director
Biomolecular Resource Center, UCSF
Box 0541, San Francisco, CA 94143
Ph: 415 514-0101x4
FAX: 415 476-7974
email: kathyi@cgl.ucsf.edu
BRC Web page: http://www.ucsf.edu/brc
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