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Long Run Big Dye Kit on 377s

Alan Wilton dna at unsw.EDU.AU
Tue May 2 04:24:52 EST 2000


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
scans).
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
signal.

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



----------------------------------------------
Alan Wilton
School of Biochemistry and Molecular Genetics
University of New South Wales
Sydney  NSW  2052
Australia

Phone	+61 (2) 9385 2019
Fax   	+61 (2) 9385 1483
Email 	a.wilton at unsw.edu.au


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