Long Run Big Dye Kit on 377s

George S. Grills grills at aecom.yu.edu
Tue May 2 12:55:10 EST 2000

Hi Alan,

We have found no difference in results between the Big Dye 1.0 and Big Dye
2.0 on the ABI377 or the ABI3700 when running pGEM controls.  We now use
the Big Dye 2.0 kit for all of our normal sequencing.  For the ABI377, we
normally run 96 lanes with porous combs and modified run conditions that we
have developed.  We use a 10 ul total reaction volume with a 1:2 dilution
of the Big Dye.  With both the old and the new Big Dye kits, we routinely
get 100% total accuracy for 800 - 900 bases and 98% total accuracy for 1000
- 1100 bases with our pGEM reaction controls.  We do see more signal
strength at longer read lengths with the Big Dye 2.0 kit compared to the
older kit, which may help with some templates that are more difficult to
sequence when we run them on either the ABI377 or the ABI3700.  Please let
me know if you would like the details of our ABI377 long read protocol.

- George

At 10:24 AM 5/2/00 +0100, Alan Wilton wrote:
>Dear Sequencers
>Has anyone tried the New ABI Dye Terminator Sequencing Kit for longer reads
>on 3700s on ABI377s?
>We find that because the smaller size fragments are extremely reduced in
>concentration (as designed) they are undetectable on a 64 lane EXcel
>Collection.  Detectable signal does not come through until 3000 scans which
>means loss of first 200 bases (Dye front still comes through at 1000
>The problem does not seems as bad if run on 36 lane gel with Standards
>Collection.  Most lanes have enough signal to detect the short sequences
>even though reduced in intensity.  Presumable it is able to detect weaker
>Has anyone else noticed this?
>What happens on 96 lane gels?
>The majority of these samples would not have been run with the full 8
>microlitres of mix per reaction.
>We find signal strength is not the problem getting long reads on 377s.  The
>loss of resolution is the problem after 700 or 800 bases.  I suggest people
>be cautions about using the new Sequencing Kit on ABI377s.  The old kit is
>likely to give better results.
>Alan Wilton
>School of Biochemistry and Molecular Genetics
>University of New South Wales
>Sydney  NSW  2052
>Phone	+61 (2) 9385 2019
>Fax   	+61 (2) 9385 1483
>Email 	a.wilton at unsw.edu.au


 George Grills
 DNA Sequencing, Genotyping, Oligonucleotide, and GeneChip Microarray
 Albert Einstein College of Medicine
 713 Ullmann Building           
 1300 Morris Park Avenue       
 Bronx, New York 10461-1602    
 Tel: (718) 430-2657
 Fax: (718) 430-8778
 E-mail: grills at aecom.yu.edu
 DNA Sequencing: http://leper1.ca.aecom.yu.edu/dnacore
 Oligonucleotide: http://sequence.aecom.yu.edu/oligo


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