Re: BAC sequencing
Deb Grove (dsg4@psu.edu)
Fri, 11 Jun 1999 12:41:32 -0400
We routinely get 500 to 800 base reads from BAC or cosmid DNA from 20 to
90K. The 90K has yielded shorter reads (400-600).
Primer length varies between 17 and 24 bases. the best reads with Tm's
greater than 60 degrees.
1.2 ug of DNA and about 30 picomoles of primer.
20 ul volume with no extra reaction mix.
Cycle Sequencing protocol:
95 for 5 min
then 100 cycles of 95 for 30 sec, 50 for 30 sec and 60 for 4 min.
The DNA comes from different labs so I am not sure what extraction
procedures they use. We have a "cosmid" protocol from one lab that provides
excellent DNA.
Deb G.
Deborah S. Grove, Ph.D.
Manager of the Nucleic Acid Facility
Biotechnology Institute
The Pennsylvania State University
210 Wartik Lab
University Park PA 16802
http://www.biotec.psu.edu/lsc/stf/naf/naf.html