We upgraded our 373 (not stretch) about 6 months ago, and definitely think
it was worth it. We always dilute the Big Dye kit with ABI's dilution
buffer 1:1, and then load 1/2 of the sample on the gel. We also do the same
thing with the dRhodamine kit, but have to load the entire sample on the gel
because the signal is a lot weaker. The reads are not all that much longer,
but are more accurate due to the more even peak heights.
As for sample prep, with the Big Dyes, the clean up is faster, because we
can get away with just a quick ethanol precipitation (60% final EtOH
concentration, no salt, room temp for 10 min, spin for 10 min, 70% EtOH wash
etc.). This is compared to the shrimp alkaline phosphatase method we were
using to clean up the "old dyes".
Best Regards,
Glenis
-- Glenis Wiebe, M.Sc. University Core DNA Services University of Calgaryphone: (403) 220-4503, fax: (403) 283-4907 e-mail: gwiebe@ucalgary.ca web: http://www.ucalgary.ca/~dnalab
mtholyst@smtpgate.bcsew.edu wrote:
> To 373 Sequencers: > > We are interested in hearing from sequencing labs who are using Big > Dye chemistry with the 373 sequencer. Specifically, was the upgrade > worth the trouble? Are your reads significantly longer, is sample > prep comparable and can you reliably use the half strength dilution > and still get readable sequence? > > We have read the ABRF sequencer study and have run test gels using a > 377 instrument so we know the upgrade is possible. We would like to > know from users if it is practical as well. > > Thanks in advance for your help, > > Terri Fletcher > Trudy Holyst > Blood Research Institute > Blood Center of SE WI > Milwaukee WI > > mtholyst@smtpgate.bcsew.edu