McClean:Cycloheximide Concentration
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Purpose
Find the minimum concentration of cyclohexamide required to fully halt translation in S. cerevisae.
Protocol
- Fill falcon tubes with 800 microL PBS + .1% tween, put on ice
- Grow up 25ml of yMM736 to mid-log phase in a klett flask.
- Take an initial 250 microL sample of the culture
- 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
- Add the given concentrations of cyclohexamide to each tube
- wait 10 minutes
- Add alpha factor to each tube, 20 microL of 10 microg/ml alpha will give 100 ng/ml alpha
- wait 1 hour
- take second time points from each tube (250 microL samples into appropriate falcon tubes)
- FACS sort samples