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1. Grow yeast cells to OD600 = 0.4 in YPD medium (pH 3.9). (Or
make alpha-factor stock solution in 0.1 N HCl.)
2. Add a-factor to a final concentration of
2 mg/ml.
3. Grow cells at 30 °C for 1 hour. Add another 1 mg/ml
a-factor. Take 10 ml of
cells and check under a microscope for bud percentage every 20 minutes.
4. When > 90% of the cells are unbudded ( ~ 2 hours after the initial addition of
a-factor ), pellet cells by centrifugation.
5. Wash twice with YPD at normal pH.
6. Grow cells in normal YPD and take samples for each time point.
7. Put samples on ice immediately.
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