TTE gels

Biopolymer Facility biopolymer at sc3101.med.buffalo.edu
Mon Jul 31 15:14:28 EST 2000


Hi Glenis,
To answer your previous question:

>No, we haven't had this problem (knock on wood).  I presume you are seeing
this with your controls as well?

Yes, it was present in all the samples on that gel, in varying degrees -
some samples looked better than others. One suggestion that was made to me
was to use the ABI LongRead basecaller. I reanalyzed with that and while
that early region was still not great to look at, the basecalling was
fairly significantly improved. I think my next step is going to be to
remake that buffer - I did have a student make it up so one only knows.
Thanks for the input...

Michelle










>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
>> >
>> >
>>
>> _/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/
>> 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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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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