Rapid Isolation of Genomic DNA from Yeast

Materials

Yeast DNA Prep Buffer

• 2 ml 10% Triton X-100
• 1 ml 10% SDS
• 1 ml 1M NaCl
• 100 µl 1M Tris pH8
• 20 µl 0.5M EDTA
• Fill to 10 ml with ddH2O

Protocol

1. Grow 10ml yeast cultures to saturation in YEPD 24C (for 2 overnights)
2. Collect the cells by centrifugation – 5min. 4,500 rpm
3. Resuspend the cell pellet in 0.5ml sterile ddH2O
4. Transfer to a 1.5ml microfuge tube and collect by centrifugation for 1 min, 10,000 rpm
5. Decant the supernatant and briefly vortex tube to resuspend pellet in residual liquid
6. Add 0.2 ml of Buffer A
7. Add 0.2 ml of phenol:chloroform:isoamyl alcolohol, and 0.3 grams of acid washed glass beads
8. Mix in the Tomy Mixer on highest speed for 4 minutes then add 0.2 ml TE pH 8
9. Centrifuge for 5 minutes, 10,000 rpm, transfer the aqueous layer to a fresh tube. Add 1 ml of 200 proof Ethanol. Mix by inversion gently.
10. Centrifuge for 2 minutes, 10,000 rpm. Discard the supernatant. Resuspend pellet in 0.4 ml of TE and 15 µl of 2 mg/ml RNaseA (DNase Free). Incubate 5 minutes at 37C. Add 8µl of 5M NH4 acetate plus 1 ml 200 proof Ethanol. Mix by inversion gently
11. Centrifuge for 2 minutes, discard the supernatant. Air dry the pellet and resuspend in 50µl TE