ABI automated sequencing using T7

Helge Weissig helgew at ljcrf.edu
Fri Jan 21 13:07:35 EST 1994


Helen Jost <mic255z at vaxc.cc.monash.edu.au> wrote:

>Dear Bionetters,
>
>We have an ABI 373A automated DNA sequencer that has been up and running for
>just under a year.  We mainly use the taq polymerase sequencing kit supplied
>by ABI and are generally happy with the results.  However, we would like to
>get back to using Sequenase/T7 polymerase for sorting out some anomalies
>that can't be resolved with taq.  With our machine, we have the capability
>to do T7 sequencing, but so far our success has been limited.  We've used the
>ABI T7 kit as supplied and have been generally disappointed with the sequence
>generated.  As sequencing has been successful with taq, I find it hard to
>believe that we're missing some critical step :-).
>
>So the question is, have people been doing routine T7 sequencing on the ABI
>machines and if so, what are their protocols ( or modifications to the one
>supplied by ABI)?  Are people generally happy with automated T7 sequencing,
>or do they just resort to manual sequencing when this is required?  Does
>anyone do automated T7 sequencing with a "home-made" kit and is the HUGE
>quantity of DNA specified by ABI really necessary for optimal results (or
>is this the cause of the problems)?
>
>Helen Jost
>Microbiology
>Monash University


Dear Helen (and dear netters),

        I have run into problems using the T7 primer and ABI AmpliTaq kit
as well, but I am not sure whether we are talking about the same thing
here.

--> Are you using labelled primers or ddNTPs? (the latter here! :))

Anyways, I mentioned the problem to the ABI rep here and also suggested to
him, that this might simply be a problem of annealing temperature. Although
the Tm for the T7 primer is theoretically considerably lower then the Tm
for the M13 primer, he did not think that that was the problem...
oh well, I haven't had the time to prove him wrong (or right for that
matter :)) but what does the net think????

The other thing I always run into is very bad sequence beyond my
approximately 200 to 500 bp long fragments (cloned into Bluescript KS II +,
so I am sequencing MCS EcoRI to KpnI, lots of Gs and Cs there). This is
independent from the primer used. Not that I am particularly interested in
that sequence, but I would be interested to solve this particular technical
problem....

any suggestions anyone?

cheers
helge



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Helge Weissig      

La Jolla Cancer Research Foundation             (619) 455 6480 x253
10901 N. Torrey Pines Rd.                  FAX: (619) 455 0181
La Jolla, CA 92034                      e-mail: helgew at ljcrf.edu
USA

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