quick DNA preps

ZEYL,CLIFFORD WESLEY,MR B7JM000 at MUSICB.MCGILL.CA
Sat Apr 8 13:48:54 EST 1995


  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




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