PCR sequencing

Leslie Johnston-Dow ldow at biosys.UUCP
Tue Oct 2 14:51:39 EST 1990


Dear Doug,
   We are doing alot of PCR sequencing.  In my own experience, 
sequencing symmetric PCR fragments can be difficult.  The things 
that have really made a difference are 1) lowering PCR primer 
amount to 5 pm each (or removing PCR primers with centicon 100) 
and 2) using Taq polymerase for sequencing and to cycle the 
sequencing reaction using a labeled sequencing primer( in our case a 
fluorescent primer).  By cycling the sequencing reaction, you will 
get a linear amplification of extension products which should give 
plenty of sequence data.  Also, with this approach you can directly 
sequence symmetric PCR fragments without any purification!  If you 
want more info just let me know....Good-luck.

Sincerely, Sandy Koepf
Applied Biosystems Inc.

I can be contacted via ldow at apldbio.com



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