I now have 7 protocols for prepping the DNA and 11 protocols for sequencing.
I am going to test several conditions* (see below) that seemed the most
variable and I'll report my results later, but for now here is a summary of
the information I received:
Preps: Mostly Alkaline Lysate protocols, or Qiagen Midi/Maxi Kits. To drop
dialyze or not to drop dialyze.*
Sequencing Protocols:
Primer length: range of 18-24mers
DNA amount: range of 0.2-5ug*
Primer amount: avg. of ~30pmol
Total volume: range of 10-40ul
Extras: Thermofidelase, DMSO, MgCl2
PCR setup: Majority used 95C for 5 min
95C for 30sec
50-55C for 20sec*
60C for 4 min
# cycles: range of 25-100*
I gave ranges when there was really no consensus in replies. If I do not
get satisfactory results (at least 400-500bp of good read) I will experiment
with the "extras" listed above.
If anyone wants any more details, please let me know.
Dina Leviten
dleviten@icos.com
ICOS, Corp.
Bothell, WA
USA