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
e-mail kraev at bc.biol.ethz.ch