Isolation of High Molecular Weight DNA from Yeast

1. For each sample grow 5 ml yeast culture to saturation in YEPD.
2. Pellet cells by spinning at ~2 K for 5 min. in tabletop clinical centrifuge. (Use 15 ml falcon tube ; orange cap)
3. Resuspend cells in 500 µls of H2O and transfer to an eppendorf tube.
4. Spin for 10k 2 min, decant the supernatent then resuspend cells in the residual YEPD by brief vortexing.
5. Add 200 µls of a solution containing :
   2% Triton X-100, 1% SDS, 100mM NaCl, 10mM Tris pH 8.0, 1mM EDTA.
6. Add 200 µls phenol / chloroform / isoamyl alcohol (25 / 24 / 1)
7. Add 0.3 g of glass beads (0.45-0.52mm). Use niffty-neato-keen bead cup on syringe.
8. Vortex for 3.5 min.(1 min. if to rescue cen. plasmid for E. coli trans.) Use Tomy® Micromixer.
9. Add 200 µls 1xTE and Spin for 7 min. at 15K.
10. Repeat phenol chloroform extraction (500 µl). Use heavy phase lock
11. Add 1 ml 100% EtOH to aqueous layer, mix by inversion.
12. Spin for 15 min. at 15 K.
13. Resuspend pellet in 400 µls 1xTE + 30 µg RNAse A (3 µl 10 mg/ml of stock) and place at 37^oC for 15 min.
14. Add 10 µls of 4M Ammonium Acetate + 1 ml of 100% EtOH + 3 µl glycogen (Roche 20 mg/ml). Mix by inversion. Put at -20C for 20 min.
15. Spin 15 min. at 15K.
16. Wash with 70% EtOH.
17. Dry pellet and resuspend in 50 µls 1xTE. (Use 5-10 µls for Southern).
    

Buffer for step 5 :
25 mls
5 mls 10 % Triton X-100
2.5 mls 10 % SDS
0.5 ml 5M NaCL
0.25 ml 1M Tris pH 8.0
0.05 ml 0.5M EDTA
16.7 mls H2O