Dear Chuck
I sequence small PCR fragments quite often, with the old and new FS kit.
We use 4ul Mix, 3.2pmol primer, 10-20ng template (We add more template if
we use the new kit to increase the signal). Total rxn volume is 20ul.
Cycling Parameters:
96C 30 sec.
50C 20 sec.
60C 2 min.
repeat 25 times
Your sequence will look dirty if the computer collects data for the full
7 or
9 hours and you don't change the stop point. This is because there are
only
~175 bases, but the analysis software will try to call bases for the
entire
run (600-800 bases). This distorts the signal. E-mail me if you have
any
questions on the stop point.
Hope this helps
Amy Tucker