Grow S. pombe cells in SD medium supplemented with appropriate nutrients to a density of approximately 1 × 107 cells/ml at 30°C. Grow S. cerevisiae cells in YPD medium to a density of approximately 1 × 107 cells/ml at 30°C.
Place the cultures on ice for 15 min just before harvesting. Collect the cells by centrifugation and wash the resulting pellet thrice with ice-cold sterilized water. Suspend this pellet in ice-cold freezing buffer containing 0.6–2.5 M sorbitol, 5–10 mM CaCl2 and 10 mM 2-(4-[2-hydroxyethyl]-1-piperazinyl)ethanesulphonic acid (HEPES; pH 7.5) to give a density of approximately 5 × 108 cells/ml.
Dispense aliquots (0.1 ml) of the cell suspension in 1.5-ml microcentrifuge tubes, slowly freeze them, and store by placing them directly in a −80°C freezer (cooling rate = ∼10°C/min).
For each electroporation, quickly thaw the frozen competent cells in a water bath at 30°C (warming rate = ∼200°C/min) and wash once with 1 ml of ice-cold 1.0 M sorbitol by centrifugation. Resuspend the final pellet in 1.0 M sorbitol to give a density of 1–2 × 109 cells/ml.
Mix the cell suspension with 0.5–10.0 ng of purified plasmid DNA and then transfer to a chilled cuvette with a 0.2-cm electrode gap. Apply a high electric pulse to the cell suspension, by using the Bio-Rad Gene Pulser II with Pulse Controller Plus.
I mmediately dilute the electroporated cells in 1 ml of ice-cold 1.0 M sorbitol and spread an aliquot (0.1–0.2 ml) on minimal selection plates. For S. cerevisiae, the minimal selection plates contain 1.0 M sorbitol as an osmotic stabilizer.
The transformant colonies appear within 4–6 days at 30°C.
