"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 phenol960 ul chloroform40 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