Adapted from the lab compendium Snowflake Yeast Protocols — Ratcliff Lab / MuLTEE (V2).
Growth
- Inoculate from the previous day’s settled fraction (see Settling selection) or from a fresh colony/stock into 10 mL fresh growth medium (YPD, per liter: 1 L water, 20 g dextrose, 20 g peptone, 10 g yeast extract, autoclaved).
- Grow with shaking for approximately 24 h, at 30 °C, 250 rpm.
Settling selection
- Vortex the culture very briefly to resuspend clusters. Do not vortex at over ~2,300 rpm.
- Transfer 1 mL of culture into a new tube to settle. Use a wide-bore pipette tip if working with macroscopic clusters.
- Settle undisturbed:
- Macroscopic populations: 30 seconds.
- Microscopic populations: 3 minutes.
- Remove the top 900 μL by eye without disturbing the settled pellet (pipette from above the 0.1 mL mark on the tube).
- Transfer 50 μL from the bottom of the remaining ~100 μL into fresh medium to start the next day’s culture (using a wide-bore pipette tip if working with macroscopic clusters).