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Star Republic: Guide for Biologists

Disrupting yeast cells

Bead mills

1. Resuspend cells in a lysis buffer.

2. Add equal volume or more glass beads.

3. Break cells using a bead mill or by vigorous vortexing. Don't let the sample get too hot. Cool on ice.

4. Let the beads settle. Transfer supernatant to a fresh tube.

5. Add more lysis buffer and run the bead mill or vortex again.

6. Combine the supernatants.

Spheroplasts

1. Yeast cell walls may be digested using zymolyase.

2. Digest 108 cells in 1 ml of 1 M sorbitol, 100 mM EDTA pH7.4, 0.1% beta-mercaptoethanol, and 100 U lyticase/zymolase.

3. Incubate at 30 C for 30 minutes with gentle shaking.

4. Centrifuge for 5 minutes at 500 g to pellet spheroplasts.

5. Disrupt spheroplasts by swelling.

See also Spheroplast.

Hot Phenol

For RNA preps, hot phenol may be used to disrupt cells.