Longest dideoxy sequencing read?

Shiao Y. Wang sywang at whale.st.usm.edu
Fri Apr 29 23:00:15 EST 1994


Steven Coon (@umich.edu) wrote:
: In article <Cp0v8v.u7 at usenet.ucs.indiana.edu>, Bill Nowatzke
: <wnowatzk at ucs.indiana.edu> wrote:

: > 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.



:     Reading 450 is actually beyond what most people can read in a gel but
: what I would do is just give it a try. If you are using USB's kit they
: provide extension mixes that will allow you to read 900 base pairs by more
: or less skipping the first part of the sequence. The range doesn't change
: but rather the sequence you are reading. If you have problems you can also
: design new primers 3-400 base pairs in and start sequencing again rather
: than having to run your gels longer. The extension mixes tend to have more
: non labeled Ntps so if you add more you can extend further. Ahigher Mg+
: concentration will also preferentially add cold rather than hot ntps in
: your reaction.

   Have you tried a gel mix called Long Ranger? We can get more readable
sequence using it compared to regular acrylamide. With two loadings, we
can reliably get about 300 - 400 using the 60 cm gel box from IBI. With
Long Ranger, we can get an extra 100 - 200.

Shiao Wang
University of Southern Mississippi




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