Hi everyone!
Have any genotypers out there dealt with this problem? When we put together
multiplexes with the FAM, HEX, NED, ROX dye set, we get a strange
"quenching" of the signal the more PCR product we load. In other words, if
I do a linear titration of PCR amounts, I get a non-linear increase in pk
hts to the point where the pk hts actually begin to decrease! This is most
easily visualized by looking at the ROX standard which steadily decreases in
pk ht as more product is loaded with it. You can see this effect even
between 0.5 and 1.0ul product! When we use the FAM, HEX, TET, TAMRA dye
set, the pk ht increase is basically linear and the TAMRA peaks stay the
same to the point (about 4ul) where salt effects begin to split the standard
peaks.
When we were using the 373's exclusively, we were dealing with the problem
by increasing PCR cycle number to enable us to load less product, but now we
are trying to use the less sensitive 377, we are back to square one. (PE
Bio says the 377 is more sensitive, but I'm looking at electropherograms of
identical samples on each machine and the 377 is 50-60% lower) Is anyone
else seeing the same thing and what are you doing about it?
Thanks very much for any ideas,
Linda
Linda Wood Ballard
Genomics Core Facility
University of Utah
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