Protoplast enzyme solution

1% BSA

1 M CaCl₂

Cellulase R10

Macerozyme R10 (Sigma)

1 M mannitol

100 mM MES-KOH (pH 5.6)

ß-Mercaptoethanol

Combine 3.9 ml of H₂O, 14 ml of 1 M mannitol, and 1 ml of 100 mM MES-KOH. Add 25 mg of Macerozyme R10 and 150 mg of Cellulase R10. Incubate for 10 minutes at 50°C. Cool to room temperature. Add 100 μl of 1 M CaCl₂, 3.5 μl of ß-mercaptoethanol, and 1 ml of 1% BSA. Filter through a 0.45-μm filter into a sterile 125-ml flask.

Prepare solution fresh ~1 hour prior to use.