Your Favorite dsDNA Sequencing Method

Bill Melchior, NCTR/FDA wmelchior at NTBTOX.NCTR.FDA.GOV
Tue Aug 11 08:37:00 EST 1992


>	I have recently isolated some positive clones from a directionally
>constructed cDNA lambda ZAP library.
...
>I am considering dsDNA sequencing.  
...
>... it produces too many GC compression artifacts),
>... would [anyone] be so kind
>as to post their favorite dsDNA sequencing method, including how they prepared
>their template and what kind of results they normally get.  I would really
>appreciate any advice (including 33-P vs 35-S vs 32-P experience).

Mike-

I have no experience with sequencing from lambda, but a few comments that might
be helpful:

0.  Since lambda is much bigger than pBR322, which I use, template preparation/
primer annealing would be different.  Someone with experience should comment
on sequencing from lambda: the advisability of subcloning into a plasmid, 
for instance.

1.  Compression artifacts do not depend on the type of sequencing, since 
they arise during the electrophoresis of newly synthesized pieces.  Whether
the template was ds or ss is immaterial.

2.  I sequence from pBR322, using the Sequenase protocol from USB, as
described in their booklet, and it works fine.  The only difference is the
method I use to prepare the template -- which seems to be a crucial step.
I can easily read about 250-280 bases using about 500 ng pBR322 template.

3.  35-S is much nicer than 33-P:  a) safer; b) longer half-life;
c) sharper bands.  As for 33-P, I have no experience -- I'm following
that thread with interest to see about others' experiences.  The advantages
over 32-P should be about the same as for 35-S, but I don't know why it
would be any better than 35-S.

________________________________________________________________________________
The opinions stated are mine, not those of NCTR or its sponsoring organizations.

Bill Melchior                                ||   "You have lawyers the way
National Center for Toxicological Research   ||    other people have mice."
Jefferson, AR  72079                         ||
(501) 543-7206                               ||   -Kenneth Duncan, English
                                             ||    Health & Safety Executive,
WMELCHIOR at NTDOC.NCTR.FDA.GOV                 ||    to US regulators
________________________________________________________________________________
The opinions stated are mine, not those of NCTR or its sponsoring organizations.

Bill Melchior                                ||   "You have lawyers the way
National Center for Toxicological Research   ||    other people have mice."
Jefferson, AR  72079                         ||
(501) 543-7206                               ||   -Kenneth Duncan, English
                                             ||    Health & Safety Executive,
WMELCHIOR at NTDOC.NCTR.FDA.GOV                 ||    to US regulators



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