Rapamycin Sensitivity Assay of Yeast

Materials

• YPD Media. See YPD Media and Agar
• YPD Plates with either DMSO or 2-200 nM Rapamycin. See YPD Media and Agar
• Yeast strains, streaked out on appropriate plate (< 1 month old)

Protocol

1. Innoculate 3 mL of YPD in a small erlenmeyer flask with one colony of yeast
2. Grow for ~8h at 24C
3. Check OD-600
4. Estimate how much to dilute yeast so that the next morning the OD is 0.3. For example, if the doubling time is 2.25h, then from 5 PM to 9 AM is 16h or 7.1 doublings, of 137 times the OD. Therefore to get an OD of 0.3, you need to innoculate at 0.3/137 = 0.00219.
5. For each flask innoculate several dilutions.
6. The next morning check OD of overnight cultures. Cultures should be between 0.1 and 0.5
7. Spin down cultures and resuspend to an OD of 3.0
8. Make 10X serial dilutions of cultures and plate 1 uL of each dilution on YPD and YPD/Rapamycin Plates
9. Grow plates at either 24 or 30C