Longest dideoxy sequencing read?
leach at mbcrr.harvard.edu
Fri Apr 29 09:29:01 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.
The time it takes u to figure it out (think man-hours and rate of
u could have made another oligo to sequence (24mer @ $2.50 a base = $60).
..... Martin Leach Email:leach at mbcrr.harvard.edu
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