"Smash and Grab" Yeast Genomic Prep

This protocol was submitted by Matt Kaeberlein.

This protocol yields DNA of sufficient quality for PCR. To obtain higher quality DNA use the zymolyase genomic prep protocol.

Required Reagants:

• Breaking Buffer
• 2% Triton X-100
• 1% SDS
• 100 mM NaCl
• 100 mM Tris-Cl, pH 8.0
• 1 mM EDTA
• Glass Beads
• Phenol/Chloroform/Isoamyl Alcohol (25:25:1) solution

Protocol:

1. Grow cells overnight in YPD.
2. Spin down 1mL of cells in eppendorf tube. Remove media.
3. Add 200 uL breaking buffer.
4. Add 200 uL phenol/chloroform/isoamyl solution.
5. Add 100-200uL glass beads.
6. Vortex 1-2 min.
7. Spin at high setting for 10 min.
8. Remove aqueous phase (top layer) to a new tube. There should be about 200uL. Discard the rest.
9. Add 1mL of cold 100% ethanol to aqueous layer.
10. Spin at high setting for 20 min.
11. There should be a small pellet at the bottom of the tube. Remove the ethanol by aspiration.
12. Resuspend the pellet in 50uL TE+RNase

I use 5uL of this prep for a 100uL PCR reaction. You can add a 70% ethanol wash to remove salts, but I've found that it's not necessary.

Glass beads can be ordered from Biospec Products Inc. Catalog #11079-105.

Phenol/Chloroform solution can be ordered directly from Sigma or you can make your own.