Dear netters,
John McDonald asked for a rapid DNA miniprep method for
screening hundreds of colonies. I've been Southern-blotting
hundreds of genotypes for population genetics studies, and ended
up using a method that combines the zymolyase and glass-bead
techniques. You need neither enzymatic cell wall digestion nor
phenol-chloroform extractions, and you get enough DNA for a couple
of Southerns, so you can even go back to colonies of interest for
further work (cloning, etc.) Since I work with ade mutants the
DNA I get has the red colour that was mentioned in a few postings
awhile ago, but it has always cut well with every restriction enzyme
I've tried.
- grow colonies overnight in 5 ml YPD in 15-ml tubes
- pellet cultures, discard supernatant and resuspend in 300 ul
water
- transfer to a 1.5 ml microtube containing 0.3 g glass beads
and 100 ul lysis buffer (see Winston glass-bead protocol
for this standard lysis buffer; to save time, instead of
weighing out dozens of aliquots of irritatingly bouncy
glass beads, mark off on a 1.5 ml tube the appropriate
volume, cut there with a razor blade and attach this
volumetric scoop to a glass Pasteur pipette or similar
handle by flaming the handle and melting it onto cut tube)
- vortex on medium (near setting 7 on our vortex mixer...)
for about 3 min; longer or more intense vortexing may
shear DNA
- add 200 ul 5M KAcetate and leave on ice for an hour
- centrifuge 5 min at max speed in a microcentrifuge
- transfer supernatant to a new tube, add 1 vol isopropanol
or 2 vol ethanol (room temp), mix, let stand at room
temp 10 min, and centrifuge for 30 seconds to pellet DNA
Cliff Zeyl
Dept. of Biology, McGill University
b7jm at musicb.mcgill.ca
