Cell cycle arrest

alpha-factor arrest

1. Grow 50 mls of yeast cells at 30C O/N.

2. Dilute into 650 mls of fresh YPD in the following morning, start from OD600=0.15, grow to OD600 0.3-0.4. Add a-factor to final concentration 1 ug/ml, grow 2h at 30C, check arresting efficiency by microscope until 90% of cells are arrested at G1 phase.

3, Wash twice with H2O. Resuspend in fresh YPD containing 50 ug/ml Pronase.

4. Grow at 30C, take 52 mls of culture every 10 minutes (from 0’ to 110’). 50 mls for ChIP, 2 x 1 ml for RT-PCR

5. Use RT-PCR to check mRNA expression and ChIP to check modifications or protein binding level.

cdc15ts arrest

1. Grow 50 mls of cdc15ts cells at RT O/N.

2. Dilute into 650 mls of fresh YPD in the following morning, start from OD600=0.1, grow at RT for 3 h.

3. Transfer cell culture to 37C and grow by shaking for 3 h. Check arresting efficiency by microscope until 95% of cells are arrested and look like dumbbell.

4. Transfer cell culture to 23C waterbath-shaker and grow by shaking, take 52 mls of culture every 15 minutes (from 0’ to 135’). 50 mls for ChrIP, 2 x 1 ml for RT-PCR

5. Use RT-PCR to check mRNA expression and ChIP to check modifications or protein binding level.