Day 0
1. Overnight yeast to be imaged.
2. Make serial dilutions - add 3.3 mL to tube A, 3 mL to tube B, 2.7 to tube C. Add sample to tube A and vortex. Take 300 uL from tube A, add to tube B, vortex. Take 300 uL from tube B, add to tube C, vortex.
Day 1
1. Label flip top tubes for each of your samples
2. Put 1 mL of appropriate overnight into each of the tubes
3. Centrifuge cells at 3600 rpm from 2 minutes at room temperature. Remove the supernatant
4. Resuspend cells in 1 mL of appropriate media
5. Add 5 uL of 0.2 M PSMF where appropriate (based on that day's experiments). PMSF is in the -20°C freezer
6. Add any drugs or vehicles where appropriate
7. Transfer to new, labeled culture tubes and shake at 30°C for 4 hours. SET A TIMER