Materials needed
- DNA extraction buffer:
- 2% Triton X-100
- 1% SDS
- 100 mM NaCl (from 5M stock)
- 10 mM Tris-HCl, pH 8.0
- 1 mM EDTA, pH 8.0
- Ref.: Harju et al, Rapid isolation of yeast genomic DNA: Bust n’ Grab, BMC Biotechnlogy 2004
- Glass beads for DNA extraction
- 1.5 ml screw-cap tubes
- 1x Tris-EDTA (TE) buffer (pH 7.4-8)
- Phenol-Chlorophorm-Isoamyl alcohol 25:24:1 (stored at 4°C)
- Ethanol 100%
- Ethanol 70%
- Recommended: RNase (stock should be 10 mg/ml)
Procedure
- Prepare 1.5ml screw-cap tubes (1 per sample):
- 0.4 g glass beads (corresponding to a volume of roughly 150 ul)
- 200 ul DNA extraction buffer
- 200 ul Phenol:Chloroform:Isoamyl alcohol
- Yeast cells (using a pipette tip, take a big chunk of cells; it is best if the cells are fresh)
- Label tubes
- Close screw caps tightly
- Load onto FastPrep (let someone show you how)
- Run the S. cerevisiae extraction protocol
- Take out tubes
- Spin down briefly in small tabletop centrifuge for a few seconds
- Add 200 ul of 1x TE
- Centrifuge at top speed for 5 min at room temperature
- After centrifugation, samples should contain an aqueous phase (top, containing the DNA), an interphase (white), and an organic phase (lipids, proteins, etc.)
- Take a clean 1.5 ml tube (no need for screw caps) for each sample, label, and add 1 ml 100% ethanol
- Transfer the aqueous phase of each sample (from step 8) to the new tubes containing 100% ethanol
- Invert samples a few times (should see nucleic acid precipitation (cloudy))
- Spin at full speed for 5 min (should see nucleic acid pellet)
- Discard the supernatant, keep the pellet
- Add 500 ul of 70% ethanol to wash the DNA pellet
- Centrifuge at top speed for 5 min
- Remove as much of the supernatant as you can, leaving as little water-ethanol as possible
- Add 50 ul 1x TE
- Recommended: add 10 ug/ml RNase (a 1/1000 dilution of 10 mg/ml stock RNase)
- Recommended (continued): at 37 C for 5 min
Author: Enrico Tenaglia
September 2018