Our sequencing service is about to convert from the old Rhodamine
terminator chemistry to the new dRhodamine terminator chemistry (dRhod
and/or BigDye). ABI's tech support recommends BigDye because of its
stronger signal strength and ability to work with smaller reaction mix
volumes (thus offset the bigger price tag). I was cautioned that dRhod has
weak signal strength problems. We've tested both with pGem control using
240 ng. in 75% reactions- 6 ul rxn. mix to final volume of 15 ul. Both
gave excellent sequence with comparable signal strengths (BigDye did have a
higher G to A ratio).
I have several questions:
1) Is dRhod inherently weaker or is there an XL upgrade effect?
2) Can dRhod handle the wide range of template types, including deviations
in quantity and quality, that University core facilities are
required to sequence? (You all have those beloved customers who always over
estimate the quantity of their templates. We love them!) Is BigDye a better
choice?
3) Is BigDye's difficulty getting through homopolymer stretches a serious
problem? Is dRhod better? We do lots of cDNA.
4) If we use BigDye, please clarify the issue of "fractions of reactions".
If you do a "half reaction" do you add 4 ul of rxn. mix to a final vol. of
10 ul or can you maintain a final vol. of 20 ul by adding buffer to keep
the proportions correct? We prefer to keep the final vol. 20 ul in order to
have more latitude for template volume (you know, for the low copy number
folks). We're concerned that too much dilution of enzyme and NTP's might
be a problem.
I welcome your advice and comments. Thanks.
Skip Vaught
DNA Sequencing Service
University of Arizona