Dir. Seq. of small PCR frag.

tucker_a at umbsky.cc.umb.edu tucker_a at umbsky.cc.umb.edu
Thu Jul 17 12:54:25 EST 1997


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



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