McClean:Cycloheximide Concentration

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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
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