Yeast Lysis by Bead Beating

1. Grow yeast to an OD 600 of 0.3-0.8

2. Spin down cells in conical tube, 3 min., 2000 rpm

3. Pour off media and resuspend pellet in remaining liquid

4. Transfer cells to screw cap eppendorf and pellet by briefly spinning in microfuge

5. Aspirate off remaining media and freeze pellets in liquid N2

6. Allow pellets to thaw on ice

7. Resuspend pellet in 2-5x pellet volume of lysis buffer + protease inhibitors

8. Add glass beads to the top of liquid level

9. Bead beat 2x 1.5 min. in master-beater, with 1 min. on ice in between beatings

10. Poke hole in bottom of tube with 21 guage needle and place tube on top of 1.5 mL eppendorf

11. Place tubes in eppendorf and spin down briefly (10 sec.)

12. Remove top tube -- recycle beads by washing out of eppendorfs with water into 50 mL conical, rinse 5X with water, rinse 2X with 95% EtOH, dry on paper towels

13. Spin bottom tube containing lysate 10 min, 14K, 4°C in microfuge

14. Transfer supernatant to new tube

15. Measure protein concentration of 0.5-5 ÁL lysate in a BioRad assay