TTE gels

Glenis Wiebe gwiebe at ucalgary.ca
Mon Jul 31 09:56:31 EST 2000


Hi Michelle,

We have been using a normal loading procedure (no water run-in, no porous
combs) with the TTE buffer for the last year, so I don't think that could be
the problem.  We haven't tried the procedure with the short gel.

Sincerely,
Glenis

--
Glenis Wiebe, M.Sc.
University Core DNa & Protein Services
University of Calgary

tel: (403) 220-4503, fax: (403) 283-4907
e-mail: gwiebe at ucalgary.ca
http://www.ucalgary.ca/~dnalab



Biopolymer Facility wrote:

> Hi George,
> I too was using your protocol, but with a few differences. I used the TTE
> buffer concentrations and modified run, however I was not using the
> membrane combs or water loading. While experimenting with these new
> conditions, I ran only small gels  and used the normal loading procedure
> with buffer in the upper chamber (not just water to start) and the mylar
> sharkstooth comb. Could these differences be a factor in the results that I
> observed?
>
> Michelle
>
> >Hi Michelle,
> >
> >There are a number of different DNA sequencing protocols from different
> >sources that use TTE.  I have seen some that have the loss of resolution
> >that you describe.  What protocol are you using?  We have developed a TTE
> >protocol (the protocol Glenis refers to) that consists of using porous
> >combs, water loading, TTE buffer, and modified run conditions.  Using this
> >method on an ABI 377, we typically achieve more than 1000 unedited bases at
> >98% total accuracy and about 900 unedited bases at 100% total accuracy with
> >standard templates such as pGEM.  In addition, we see significant
> >improvement in read length with many difficult-to-sequence templates, such
> >as GC rich templates.  We can get clean reads from the start of the
> >sequence.  Please let me know if you would like me to send you details of
> >the protocol.
> >
> >- George
> >
> >
>
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> Biopolymer Facility
> Roswell Park Cancer Institute
> Buffalo, NY   14263
> Phone: (716) 845-8032
> Fax: (716) 845-7621
> Email: biopolymer at sc3101.med.buffalo.edu
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> ---


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