Rapamycin Sensitivity Assay of Yeast
Revision as of 18:21, 14 October 2009 by Davebridges (Talk | contribs) (added notes about dilutions and incubations)
Revision as of 18:21, 14 October 2009 by Davebridges (Talk | contribs) (added notes about dilutions and incubations)
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
- Innoculate 3 mL of YPD in a small erlenmeyer flask with one colony of yeast
- Grow for ~8h at 24C
- Check OD-600
- 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.
- For each flask innoculate several dilutions.
- The next morning check OD of overnight cultures. Cultures should be between 0.1 and 0.5
- Spin down cultures and resuspend to an OD of 3.0
- Make 10X serial dilutions of cultures and plate 1 uL of each dilution on YPD and YPD/Rapamycin Plates
- Grow plates at either 24 or 30C