McClean:Cycloheximide Concentration

Purpose

Find the minimum concentration of cyclohexamide required to fully halt translation in S. cerevisae.

Protocol

1. Fill falcon tubes with 800 microL PBS + .1% tween, put on ice
2. Grow up 25ml of yMM736 to mid-log phase in a klett flask.
3. Take an initial 250 microL sample of the culture
4. Alloquot 2ml of culture into 10 different test tubes and put on rollers, labeled as follows:
   • control (no cyclohex no alpha)
   • 100 microg/ml cyclohexamide only (no alpha)
   • 1 mg/ml cyclohexamide
   • 100 microg/ml cyclohexamide
   • 10 microg/ml cyclohex
   • 1 microg/ml
   • 100 ng/ml
   • 10 ng/ml
   • 1 ng/ml cyclohex
   • 0 ng/ml cyclohex
5. Add the given concentrations of cyclohexamide to each tube
6. wait 10 minutes
7. Add alpha factor to each tube, 20 microL of 10 microg/ml alpha will give 100 ng/ml alpha
8. wait 1 hour
9. take second time points from each tube (250 microL samples into appropriate falcon tubes)
10. FACS sort samples