Karissa Milbury
posted about 1 month ago
A.K.A. Point Mutation 🔬

"Quick and dirty" S. cerevisiae gDNA prep protocol

This protocol served me well through my PhD and postdoc years. Dilute the final product at least 1/10 for use in PCR.

  • Spin down 50-100 uL packed volume of yeast cells
  • Resuspend in 200 uL TE + 1% SDS and move the cells to a 1.5ml screw-top tube if possible
  • Add 200 uL of glass beads (eyeball this: just pour in beads until the level of beads matches the level of liquid, and the total volume looks ~400uL)
  • Add 400 uL of PCI (Phenol:Chloroform:Isoamyl alcohol at 25:24:1). Master mix for 5 samples:
  • 1000 ul phenol
  • 960 ul chloroform
  • 40 ul isoamyl
  • Screw on tube tops TIGHTLY and vortex at max speed (in a fume hood!!) for 10 min. If you don't have screw top tubes, use a plastic top fastener. Having one of these pop open while vortexing sucks
  • Spin down at 13,000rpm at room temp for 10 min
  • Move the aqueous layer to a fresh tube (discard the rest) and add 1ml 100% ethanol to it; invert to mix
  • Spin at 13,000rpm at room temp for 10 min
  • Dry the pellet by pouring off the ethanol and leaving the tubes open, face-down, on a kimwipe. I usually leave them 20-30 min but depends on the humidity of your lab
  • Resuspend in 200 uL TE and store at -20C
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