Sequencing large banks

Sandy Beeser abeeser at utmem1.utmem.edu
Fri Oct 25 22:26:35 EST 1996


  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.

Alexander Beeser
Grad student 
University of Tennessee, Memphis



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