Fluorescent Sequencer Questions

[lsimon at ALNUS.FOR.ULAVAL.CA]

Fluorescent Sequencer List Members et al.,

>Topic 1: Sequencing with PCR products as templates.
>
>1. What kind of success have you had with sequencing PCR products?
>2. If you have had "good" success with PCR product sequencing, how's about
>   passing on the protocol for PCR product isolation and the sequencing
>   conditions?
>
I had good success with the ABI Dye Terminator using their exact protocol, when 
applied to my PCR products. I clean my PCR products by centrifugal 
ultrafiltration ( Centricon-30 or Millipore's Ultrafree TTK), taking care to 
rince the retentate 3 times with 'PCR' water. This is the same procedure that 
gave me good results in manual sequencing of asymetric PCR. My PCR products are 
from 300 to 900 bp. My only problem so far has been to reliably get rid of 
excess Dyes: I tried the suggested ABI protocol with Bio-Rad's spin columns (Bio 
Spin 30) and speed-vac of the eluate, but only 1 in 3 gets very clean.

Since I don't have a lot of experience with the ABI sequencing of other 
templates, I can't compare...but I'm happy with reading approx. 250 nt. with 
1% ambiguities!
Luc Simon

Centre de recherche en biologie forestiere
Faculte de foresterie et geomatique
4145 Abitibi-Price
Universite Laval
Sainte-Foy, G1K 7P4

(418) 656-5496
(418) 656-3551 fax

lsimon at alnus.for.ulaval.ca