Grow the cells overnight with vigorous aeration in 50 ml of YPD (1% yeast extract, 2% bactopeptone, and 2% dextrose) to a concentration of about 3 × 107 cells/ml and harvest.
Wash the cells successively with 20 ml of sterile water and 20 ml of 1 M sorbitol by resuspension, followed by 5-min spins. Resuspend them in 20 ml of SCEM [1 M sorbitol, 0.1 M sodium citrate (pH 5.8), 10 mM EDTA and 30 mM 2-mercaptoethanol], add 1,000 U of lyticase, and incubate at 30°C with occasional inversion.
After spheroplasting, measure the decrease in the OD800 of a 10-fold dilution of spheroplasts in water. Harvest the spheroplasts for 3–4 min when the spheroplasting proceeds to 90% (∼15–20 min).
Gently resuspend the spheroplasts in 20 ml of 1 M sorbitol by using a 1-ml pipette and pellet for 3–4 min. Then, gently resuspend them in 20 ml of STC [1 M sorbitol, 10 mM Tris-HCl (pH 7.5) and 10 mM CaCl2] and pellet again for 3–4 min. Resuspend this pellet in 2 ml of STC.
Mix aliquots (100 µl) with plasmid DNA and carrier DNA (calf thymus or E. coli) added to a total of 5 µg of DNA in <10 µl.
After 10 min at room temperature, add 1 ml of PEG [10 mM Tris-HCl (pH 7.5), 10 mM CaCl2 and 20% PEG 8000; filter-sterilized], gently resuspend the spheroplasts, and harvest them for 4 min after another 10 min.
Resuspend the pellet in 150 µl of SOS (1 M sorbitol, 6.5 mM CaC12, 0.25% yeast extract and 0.5% bactopeptone; filter-sterilized) and leave at 30°C for 20–40 min. Dilutions of the spheroplasts are made in the same medium.
Add 8 ml of TOP [1 M sorbitol and 2.5% agar in selective SD medium (0.67% yeast nitrogen base and 2% glucose)] kept at 45–46°C. Invert the tube quickly several times to mix and plate the suspension immediately on selective SORB plates (SD plates containing 0.9 M sorbitol and 3% glucose).
