Re: DNASeq: chemistry option

Scottie Adams (sadams@northnet.org)
Thu, 11 Feb 1999 19:29:29 -0400

At 10:36 AM 2/11/99, Martin Pentony wrote:
>I'm also interested in comments on the DYEnamic ET terminator. At present
>we are using BigDyes. I had some problems with secondary structures. I
>tried raising the extension temperature to 70 but the reactions failed.
>This had worked before with Amersham Rhodamine kits. Have the ABI kits a
>lower temperature tolerance? My impression is that the amersham ET
>terminators work as well as the BigDyes and are better for difficult
>templates like secondary structure and poly A or poly T. Comments from the
>experienced would be most welcome. Thanks

Martin

I can't tell you about the ET dye terminators- but I have been
using the Amersham "old" rhodamine dyes with the Thermosequenase II on an
old 373S and have been quite happy with them. I used them recently on the
ABRF DNA Sequencing Research Committee's Difficult (DSRC)template - Sasha-
which is very GC rich and I got results which were as good if not better
than those obtained with Big Dyes on a 377.

Hopefully, if enough labs participate in the DSRC Difficult
Template study - we may have some answers for you. :-) YES- this is a
big hint!!!
Submit those results for Sasha and Lunatic and don't forget to send some
pGEM results, too. We would all like to know more about the different
chemistries and machines available. The results will be presented at
ABRF'99 and posted on the web asap.

Scottie

Scottie Adams aka Pamela Scott Adams

Molecular Biology Core Facility
Trudeau Istitute
100 algonquin Avenue
Saranac lake, NY 12983
Phone: 518-891-2237
Fax: 518-891-5126
Email: sadams@northne.org
http://www.trudeauinstitute.org