Longest dideoxy sequencing read?

Martin Leach 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
money/hour)

u could have made another oligo to sequence (24mer @ $2.50 a base = $60). 

Martin.

-- 

.....          Martin Leach                Email:leach at mbcrr.harvard.edu 
   _|____      Dept. of Pharmacology       Phone: (617) 638-5323        
   / o  /      Boston Univ. School of Med. Fax:   (617) 638-4329         
 _/  |-/__==/  80 E. Concord St. (L603)
(BULLDOZER) \_ Boston MA 02118            "Not the old underpants on your
               USA                           head.....WIBBLE" -BLACKADDER 
p.s. try BioMOO (virtual biology on the internet - telnet
bioinfo.weizmann.ac.il 8888)
 



More information about the Methods mailing list