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 (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
Purdue University Genomics Core Facility