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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