Protoplast Transformation

Materials

• P buffer
• 10.3% sucrose
• 1 g polyethylene glycol 1000 (PEG)
• R5 or R2YE plate

Instructions

1. Follow the instruction in Protoplasts Formation, Regeneration and Transformation
2. Add 3.85 ml P buffer to 1 g PEG sterilised.
3. Add 200 µl PEG-P buffer mixture and a DNA solution to 50 µl of the protoplast suspension.
4. Plate it onto R5 or R2YE.
5. Incubate the plate at 298-30ºC overnight.
6. Overlay 1 ml sterilised water containing nalidixic acid and an appropriate antibiotic.