( 1. Inoculate 50 ml of YEPS...)Ustilago maydis

1. Inoculate 50 ml of YEPS with a primary culture of Ustilago maydis and grow cells overnight at 30 C to a cell density of O.D.₆₀₀ of 0.5 to 0.7. 2. Harvest by centrifugation at 3,000 rpm for 5 min at room temperature. 3. Wash one time with 25 ml SCS. 4. Digest the cell walls by resuspending cells in 2 ml filter sterilized SCS containing 3 mg/ml Novozyme-234, incubate until 20 to 50% of protoplasts have been formed (observe by microscope), then add 25 ml 1 M ice-cold sorbitol. 5. Centrifuge for 7 min at 2,300 rpm at 4 C. 6. Wash three times with 25 ml ice-cold 1 M sorbitol. Centrifuge for 7 min at 2,300 rpm at 4 C. 7. Resuspend in 1 ml ice-cold 1 M sorbitol (density: 10⁷ to 10⁸ protoplasts/ml). Keep on ice.

Electroporation of cells:

1. Add 1-2 l pNEBUH DNA or 10-20 l pSMUT DNA (1 g/l) to 400 l of protoplasts. Homogenize by gently mixing with pipette several times. Transfer mixture into a prechilled cuvette.
2. Wipe moisture from the cuvette and insert the cuvette into the device.
3. Electroporation:
   
   Mode Prokaryotes O Voltage (V) 480 V Time constant (T) 5 ms
   
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1. Ustilago maydis