'Half BD' big dye diluent

George S.Grills grills at aecom.yu.edu
Wed Nov 15 17:15:29 EST 2000


Hi Kevin,

We have tried the ABI 5X dilution buffer, the GenPak halftermBD dilution
buffer, and a homebrew dilution buffer stock of 4 mM Tris pH 9.0 + 10 mM
MgCl2.  We had similar results with all of them.  For our production
384-well plate sequencing, we typically use 0.5 ul of BigDye terminator +
3.5 ul of homebrew dilution buffer stock (4 mM Tris pH 9.0 + 10 mM MgCl2)
in a total reaction volume of 10 ul.  In controlled experiments, we found
no reduction in read length using any of this dilution.

An analysis of the effects of different protocols on sequencing quality can
be found on the ABRF DNA Sequencing Research Group 1999 web poster at
http://www.abrf.org/ABRF/ResearchCommittees/dsrcreports/dsrg99/dsrg99.pdf.

Another good source to compare the results of using dilution buffers and
other modified sequencing protocols is the ABRF DNA Sequencing Research
Group NeverEnding Study (NES), a searchable, on-line database resource that
aims to assist DNA sequencing laboratories in performance evaluation,
quality control and decision making.  NES can be found at
http://nes.biotech.cornell.edu/nes.
 
- George


At 03:14 PM 11/15/00 +0000, you wrote:
>Has anyone tried the diluents on the market that you can add to BigDye
>to reduce the amount of sequencing kit you use?
>
>Are they any good?
>
>How little BD kit can you use and does it reduce read lengths?
>
>Thanks
>
>Kevin
>


________________________________________________________________

 George Grills
 Director
 DNA Sequencing, Genotyping, and GeneChip Microarray Facilities
 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
________________________________________________________________



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