Longest dideoxy sequencing read?

Bill Nowatzke wnowatzk at ucs.indiana.edu
Fri Apr 29 08:10:07 EST 1994


I am trying to read about 800 bp of a G+C rich stretch of DNA cloned into
a pBluescript. To date I have read 450 bp by staggering my loads (run for
10 h at 1600 V). I am just wondering how promising the chances are to
read much farther with dideoxy sequencing. If I run for say 15 h can I
expect to read 600bp? Do I need to increase dNTP concentrations for this.
I tried a 15h run yesterday (haven't developed the film yet) and I had
some funky gel problems such as the wells squishing up into the comb. The
longer it ran the more the gel seemed to expand. I did sweep a Geiger
over the dried gel and there seemed to be dismal label in the long run
lanes. I am working on running a Licor (fluorescent tag?) sequence that
is suppose to be able to read 900 bp. I would be interested in comments
on this also. I have a protocol for the rxns, but I heard that it may
require special template treatment (Quiagen column purification?) etc.
Thanks for your comments, Bill.



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