PCR Sequencing

[Doug Prasher]

Fellow Netters,

If you are directly sequencing PCR products, I need your advice.

We are trying to sequence PCR products of 400bp and 750bp in length 
and having almost no success.  We have tried changing a number of variables
none of which improve the situation.  Our M13 control reactions done
in parallel ALWAYS work.

OUR STANDARD CONDITIONS are:  0.6 pmol ds template, 6 pmol primer
(also used in PCR), boiled 10 minutes, snap frozen in liquid nitrogen.
Thawed on ice and then sequenced using Sequenase.

THE GENERAL RESULT is:  No sequence ladder (or extremely faint) but 
there is a fairly intense band in all four lanes at a position 
in the sequencing gel approximating the full-length fragment.  
On some gels, a short sequence ladder is observed just below this 
intense band, which we intrepret as termination at the end of the fragment.

We have assumed the problem results from a low concentration of the 
template-primer complex in the labelling reaction.  We believe there 
is enough nucleotide present in the labelling reaction such that
the Sequenase makes the complexes completely double-stranded before
the dideoxys are added in the termination reaction.

We have tried the following variables, individually of course,
 with no improvement:

1) 4 pmol ss template (via assymetric PCR) instead of 0.6 pmol ds template.
2) Increasing sequencing primer to 40 pmol.
3) Changing the thawing conditions: 
	i) leave on ice 90 min
	ii) place in a -20C block, let warm to 7C or 15C.
4) Dilute labelling mix: 1:5 is normal, 1:20, 1:75, 1:300.
5) Shorten labelling reaction time at RT and at 0 C.

PLEASE SEND YOUR COMMENTS.  IF REQUESTED I WILL SUMMARIZE AND REPOST.

Douglas Prasher
Woods Hole Oceanographic Inst
Woods Hole, MA

prasher at hyatt.whoi.edu