Cycloheximide Experiment

1. Grow cells up overnight in 50ml culture
2. In morning of second day check OD of cells. The OD600 should be about 1.0. Remove 10ml as time 0 and added to 50 ml conical tube containing 100 microliter of 100mM sodium azide (NaN₃). This is to instantly kill the cells.
3. Add cycloheximide to remaining 40 mls to a final concentration of 100mg/ml.

• Make 10mg/ml stock in water (100x)

4. Add 100ml of NaN₃ (final concentration 1mM) to each conical tube before harvesting cells

• Use stock at 100X (100mM).
• NaN₃ molecular weight at 65.01

5. Remove 10 ml of cells at specified time points. Centrifuge. Remove supernatant and shock freeze. Store at –80⁰C

Good starting time course:

• 10’, 30’, 100’, and 300’ minutes