I noculate the yeast strain into 5 ml of liquid medium (2x YPAD or synthetic complete [SC] selection medium) and incubate overnight at 30°C. Place a bottle of double-strength YPAD broth (2x YPAD) and 250 ml culture flask in the incubator as well.
Determine the titer of the yeast culture by measuring the OD600 of a solution of 10 µl of the cells added to 1.0 ml of water in a spectrophotometer cuvette. For many yeast strains, a suspension containing 1 × 106 cells/ml will give an OD600 of 0.1.
Transfer 50 ml of the pre-warmed 2x YPAD to the pre-warmed culture flask and add 2.5 × 108 cells to give a density of 5 × 106 cells/ml. Incubate the flask on a rotary or reciprocating shaker at 30°C and 200 rpm. (Note: It is important to allow the cells to complete at least 2 divisions. Transformation efficiency remains constant for 3 to 4 cell divisions).
When the cell titer is at least 2 × 107 cells/ml, which should take about 4 h, harvest the cells by centrifugation, wash the cells in 25 ml of sterile water, and wash again in 1 ml of sterile water.
Add water to a final volume of 1.0 ml and vigorous vortex-mixing to resuspend the cells. Pipette 100 µl samples (∼108 cells) into 1.5-ml microcentrifuge tubes, one for each transformation, centrifuge at top speed for 30 s, and discard the supernatant.
Add 360 µl of transformation mix, consisting of 240 µl PEG 3350 [50% (w/v)], 36 µl LiAc (1.0 M), 50 µl boiled single-stranded DNA (2.0 mg/ml), and 34 µl plasmid DNA plus water, to each transformation tube and resuspend the cells by vigorous vortex-mixing.
I ncubate the tubes in a 42°C water bath for 40 min. [Note: The optimum time can vary for different yeast strains].
Microcentrifuge at top speed for 30 s and remove the transformation mix with a micropipette. Pipette 1.0 ml of sterile water into each tube, stir the pellet with a micropipette tip, and vortex.
Plate appropriate dilutions of the cell suspension onto SC selection medium.
