Table 2.

Original protocol for the lithium method developed by Ito et al.²

Grow the yeast cells aerobically on 100 ml of YPD medium at 30°C with reciprocation. At the mid-log phase, harvest the cells by centrifugation, wash once with TE [10 mM Tris-HCl (pH 8.0) and 1.0 mM EDTA] and suspend in TE to a final concentration of 2 × 108 cells/ml. To a 0.5-ml portion of this cell suspension, add an equal volume of 0.2 M metal ions (LiAc). After 1 h at 30°C with shaking (140 rpm; stroke, 7.0 cm), incubate 0.1 ml of the cell suspension statically with 15 µl of a plasmid DNA solution (670 µg/ml) at 30°C for 30 min. Add an equal volume of 70% PEG 4000 dissolved in water and sterilized at 120°C for 15 min and mix thoroughly on a vortex mixer. After standing for 1 h at 30°C, incubate the suspension at 42°C for 5 min. I mmediately cool the cells to room temperature, wash twice with water, and suspend in 1.0 ml of water. For selecting the yeast transformants, directly spread 0.1 ml of the cell suspension on selective solid medium.