alpha-factor synchronization (arrest-release)
- Use fresh plate of cells to get reproducible results.
- Inoculate a 50ml culture and grow overnight.
- Next morning centrifuge the culture, wash with 1 volume YPD and resuspend pellet
in 220 ml YPD + α-factor (10 μg/ml).
- Incubate the culture for 2:30 h and check arresting efficiency by microscope until
90% of cells are arrested at G1 phase.
- Centrifuge the culture and wash with 1 volume YPD without α-factor.
- Resuspend in 220 ml of medium and incubate for 2 hours.
- Take 30 ml samples each 20 minutes.
- Take 0.3 ml sample for FACS analysis.
- Crosslink with 1% formaldehyde 30min at RT each sample.
- Quench formaldehyde with 125mM glycine 5min at RT.
- Spin cells and resuspend in 20ml of ice cold TBS and repeat this washing step.
From that step samples must stay ice-cold.
- Centrifuge and resuspend cells in 800 lysis buffer.
- Add this mixture to 500µl of acid washed glass beads and vortex on a bead
mill 10 min at 4C
- Collect samples by puncturing the lid and the bottom of the tube with a needle
and centrifuge 1min at 1000rpm in a 15ml falcon tube
- Shear chromatin by sonication: 2 X 15min at maximum output with 5min on ice
in between using the Bioruptor Tween sonicator.
- Centrifuge 15min at max speed and take the supernatant.
- Inoculate a 50ml culture and grow overnight.
- Next morning centrifuge the culture, wash with 1 volume YPD and resuspend pellet
in 220 ml YPD + α-factor (10 μg/ml).
- Incubate the culture for 2:30 h and check arresting efficiency by microscope until
90% of cells are arrested at G1 phase.
- Centrifuge the culture and wash with 1 volume YPD without α-factor.
- Resuspend in 220 ml of medium and incubate for 2 hours.
- Take 30 ml samples each 20 minutes.
- Take 0.3 ml sample for FACS analysis.
- Crosslink with 1% formaldehyde 30min at RT each sample.
- Quench formaldehyde with 125mM glycine 5min at RT.
- Spin cells and resuspend in 20ml of ice cold TBS and repeat this washing step.
From that step samples must stay ice-cold.
- Centrifuge and resuspend cells in 800 lysis buffer.
- Add this mixture to 500µl of acid washed glass beads and vortex on a bead
mill 10 min at 4C
- Collect samples by puncturing the lid and the bottom of the tube with a needle
and centrifuge 1min at 1000rpm in a 15ml falcon tube
- Shear chromatin by sonication: 2 X 15min at maximum output with 5min on ice
in between using the Bioruptor Tween sonicator.
- Centrifuge 15min at max speed and take the supernatant.