'Half BD' big dye diluent

Phillip San Miguel pmiguel at purdue.edu
Mon Dec 18 12:34:59 EST 2000


Joyce Bennett wrote:

> We have an ABI 3700. I tested the halfBD diluent using BigDye terminator
> version 2 reaction mix and pgem with m13 forward primer.  In my hands, using
> 3 ul of reaction mix and 3 ul of halfBD resulted in over 600 bp reads. Using
> 2 ul of each decreased the read length to 300 bp. I would suggest  getting a
> free sample and trying it in your lab.
> [...]

I use Bruce Roe's method[1] (actually a slight variation on it) to do 1/8th reactions on the 3700. I
average over 500 basepair reads (counting failures). In my hands the critical factors are using 5%
DMSO (final volume) and making sure there is no alcohol contamination of the DNA preps. The last
point is critical I think. A little ethanol contamination of your DNA has little effect on full
reactions--but for 1/4th or more you count on your enzyme being stable for extra themal cycles.
Also, I use the original big dye mix, not the version 2. Not sure if you can dilute version 2 as
much.
Phillip SanMiguel
Purdue University Genomics Core Facility



[1] http://www.genome.ou.edu/big_dyes_plasmid.html


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