I posted a message this week about out of phase A/C and G/T peaks on my
ABI 373 sequencer. My Amersham rep pointed me in the
direction of one of their scientists who easily diagnosed the problem.
The A and C are fluorescein and the G and T are rhodamine dyes. There is
a two charge difference between the two and the differential migration of
these is compensated for by the mobility files. Running a new matrix
file does nothing to repair this problem. The Long Ranger gel recipe I
was using ( double checked before I ever tried to use the stuff by
confirming the method over the phone with FMC) does not analyze correctly
with the 6% or the 4% primer mobility files. Different mobility files
screwed the data up in different places. Alignment was better early to
mid with the 6%, better later with the 4%. FMC then told me they now
have better parameters, %Ranger, TEMED, APS etc. for the 373 and are
faxing the protocol.
I had started using the Long Ranger with terminators and have not
noticed as severe a problem, but caution to all who are using new gel
chemistries. Amersham's new primer system is coming with it's own
mobility files, thanks..... Thanks to Jim Tomfohrde of Amersham for
finding the solution to a month of torture, when ABI, I assume unaware,
had no idea it was the Long Ranger.
Carla Drebing
Univ of CO Health Sciences Center
Denver, CO 80262
303-399-8020 ex2722
drebingc at essex.hsc.colorado.edu
