prasher at HYATT.WHOI.EDU
Fri Sep 28 12:29:57 EST 1990
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.
Woods Hole Oceanographic Inst
Woods Hole, MA
prasher at hyatt.whoi.edu
More information about the Methods