From: mcada002@mc.duke.edu
Date: Thu, 23 Mar 2000 15:09:10 -0500
Subject: Big Dye, V.2?
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Has anyone used the Big Dye version 2 for DNA sequencing? Would you
please
share you observations?
Thanks,
Millie McAdams/HHMI Biopolymer Facility @ DUMC
mcada002@mc.duke.edu
919-684-2652
Dear Millie,
I run a sequencing facility in Manchester, England and have done some
provisional work with version 2 big dyes.
One tentative observation is that absolute signal strenghts tend to be
higher than version 1 chemistries.
Infact, the main theoretical difference is that read lengths are
potentially longer with version 2 big dyes, owing to a higher ratio of
dNTP's to ddNTP's', which augments synthesis of longer extension
products and consequently strengthens the signal at the 3' end of the
sequence in particular.
This is beneficial for reads derived from 48cm plates on 377's,
particularly if you resort to newer protocols designed to maximise read
lengths from 377 gels, viz. running with Tris TAPS buffer rather than
the usual Tris Borate mix.
Hope this helps and let me know if you require any more details.
Best wishes,
Laurence S. Hall.
*****************************************************************************
Laurence S. Hall,
Experimental Officer,
School of Medicine,
Stopford Building,
University of Manchester,
Oxford Road,
Manchester M13 9PT,
ENGLAND.
Tel. : +44 (0)161 275 4867.
Fax : +44 (0)161 275 5272.
E-Mail :Lhall@man.ac.uk
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