Rhodamine sequencing for ABI

Dom Spinella dspinella at chugaibio.com
Thu Jul 31 13:08:59 EST 1997


Andrey Shaw writes:

> What's the experience at there with the new ABI rhodamine sequencing
> kit. We tried one gel, and it look FANTASTIC. The actual gel image
> looked preety bad with almost no signal, but the sequence files were
> beautiful. Has anyone had any problems with this kit?
> 
> andrey shaw

Andrey: 
Our Sequencing core facility has switched entirely to the new rhodamine
terminators.  They work very well, but there are a few caveats.  First,
the reason they are superior is because there is less cross-talk between
the 4 different dyes, i.e, their emmision spectra has less overlap. 
Thus, there is less electronic subtraction needed (which is what is done
when you make a "matrix" as ABI calls it) which translates into better
signal-to-noise ratios.  The problems that you noted on looking at the
gel image is that rhodamine dyes are pretty dim (i.e their quantum yield
is low) -- this has been known for a long time in the flow cytometry and
fluorescence microscopy worlds.  Hence, your signal intensity is lower
than with the old dyes.  You can also see the difference when you look
at the signal intensity numbers on the electropherograms -- they rarely
get much over 100, whereas the old FAM, JOE, ROX, and TAMRA dyes could
get over 300 on a good day. You can compensate for this somewhat by
simply increasing the number of cycles in the cycle sequencing protocol
and/or by loading more of the sample.  I also wonder whether increasing
the laser intensity would help, but we haven't tried this. However, like
you, we also observe great data and very long lengths of read even when
the gel images are quite faint.  So I wouldn't worry about the gel image
if your analyzed data are giving you the results you need.  As long as
you can see enough of the gel image to ensure proper lane tracking (now
there's something to really complain about in ABI sequencers!), does it
really matter that the image is faint?  Cheers  -- D.G. Spinella



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