Sequencing large banks

[Alexander Kraev]

In article <abeeser-2610961126350001 at tcooper2.utmem.edu>,
abeeser at utmem1.utmem.edu (Sandy Beeser) wrote:

>   Has anyone had good experience sequencing small amounts ( say 100 bp
> then its off to the database) from really large plasmids using cycle
> sequencing ?  We have a Yep13 chromosomal bank ( size 17-32 kb) that I
> can't get any sequence from using promega's fmol system with direct
> incorporation, although it will sequence pBR322 with no problem.  Should I
> just scrap direct incorporation and go for end-labelling the oligo ? 
> Techincal support at promega said size should not matter, although they
> said that it would require some optimisation with regards to
> template/primer concentrations as well as cycling parameters.  Any help
> will be appreciated.


SIZE DOES MATTER, because you need your fmols to be present for a
readable result.  You should scrap direct incorporation and go for a 33P
labelled primer with a Tm of 70 C and possibly use a Phosphorimager
instead of an X-ray film.  And make sure you have at least 1 fmol of
template in the reaction. Good luck.

-- 
Alexander Kraev, PhD
Biochemie III, ETHZ Zurich
Phone 41-1-632-31-47
Fax 41-1-632-12-13
e-mail kraev at bc.biol.ethz.ch