Longest dideoxy sequencing read?

Steven Coon at umich.edu
Fri Apr 29 10:25:12 EST 1994


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.


USB gives away for free (at least they used to) a huge manual for
sequencing with a video that has EVERYTHING you can imagine about
sequencing. BUY it if you  have to. It is well worth it!!! 

Good luck,

-- 
Steve Coon               *    Life in the fast Lane :        ---    __o    
           
                         *                               -- --    _ \ \_~  
          
sdcoon at umich.edu         *                                 ..... (_)/ (_) 
University of Michigan   *
 School of Dentistry     *    Faster, Faster the lights are turning red !
 
                               



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