TCA Precipitation for S. cerevisiae
1. Grow 5 ml overnight/stationary phase culture
2. Dilute 1:10-20 in rich media and grow for 2-3 hrs at 300C (concentration of cells should be around OD600 = 0.5-0.8)
3. Spin down cells, resuspend in 1 ml ice-cold water, transfer to Eppendorf tube
4. Pellet cells, remove supernatant
5. Resuspend pellet in 500 µl 20% TCA
6. Add glass beads (~ 100 µl), vortex 30 min at 40C
7. Transfer lysate to new tube, wash the glass beads with 200 µl 5% TCA and add to the tube containing the rest of the lysate (the tube with glass beads is discarded)
8. Centrifuge 14.000 rpm for 15 min at 40C
9. Wash pellet with 1 ml 70% EtOH (it is best to resuspend the pellet, which usually requires some effort)
10. Spin down 5 min 14.000 rpm and remove as much EtOH as possible (be careful not to lose parts of the pellet)
11. Resuspend pellet in 250µl 2 x Laemmli sample buffer by pipetting (will probably turn yellow due to acidic TCA)
12. Add unbuffered Tris-base (1M) until sample turns blue
13. Boil 5 min
14. Spin down insoluble components, load ~ 20 µl on gel
2. Dilute 1:10-20 in rich media and grow for 2-3 hrs at 300C (concentration of cells should be around OD600 = 0.5-0.8)
3. Spin down cells, resuspend in 1 ml ice-cold water, transfer to Eppendorf tube
4. Pellet cells, remove supernatant
5. Resuspend pellet in 500 µl 20% TCA
6. Add glass beads (~ 100 µl), vortex 30 min at 40C
7. Transfer lysate to new tube, wash the glass beads with 200 µl 5% TCA and add to the tube containing the rest of the lysate (the tube with glass beads is discarded)
8. Centrifuge 14.000 rpm for 15 min at 40C
9. Wash pellet with 1 ml 70% EtOH (it is best to resuspend the pellet, which usually requires some effort)
10. Spin down 5 min 14.000 rpm and remove as much EtOH as possible (be careful not to lose parts of the pellet)
11. Resuspend pellet in 250µl 2 x Laemmli sample buffer by pipetting (will probably turn yellow due to acidic TCA)
12. Add unbuffered Tris-base (1M) until sample turns blue
13. Boil 5 min
14. Spin down insoluble components, load ~ 20 µl on gel