Adapted from this protocol

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• 1M Lithium Acetate (sterile) (LiAc) 10.2 g in 100 ml dH20 and autoclave.
• 100 mM Lithium Acetate (sterile)(Dilute the 1M with ddH2O)
• Glycerol (sterile)

1. Day 1: Grow up your yeast strain in the appropriate medium overnight. If YPD medium is used, grow 2 ml for every 100 ml intended to inoculate the next day. If you are growing a yeast strain with a plasmid that needs auxotrophic selection, grow in appropriate YNB based selection medium about 20 ml for every 100 ml intended to inoculate the next day. Some yeast strains in selection media may take 2-3 days to reach saturation. You should grow just to the point of saturation.

2. Day 2: Inoculate for example 250 mL YPD which is enough for about seventy-five 100 µL aliquots of frozen competent cells. Scale up or down as needed. Inoculate to an initial OD640 to about 0.5 * 1E7 cfu/mL. The relationship between OD640 and cfu/mL depends very much on both yeast strain used and type of spectrophotometer. If you use a GENESYS20 spectrophotometer and a CEN.PK yeast strain, we have found that and OD640 of 0.17 corresponds to 0.5 * 1E7 cfu/mL. Let the cells grow at least two generations to an OD640 of at least 0.68 if you started from 0.17. This takes about four hours at 30°C for CEN.PK, but may take longer for other strains.

3. Spin down cells (? g for 5 min), pour off the supernatant.

4. Resuspend in 40% of starting culture volume (i.e 100 ml if you grew up 250 mL culture) of a 100 mM solution of LiAc. Mix well, use vortex if necessary.

5. Spin down cells (? g for 5 min), pour off the supernatant.

6. Resuspend in 20% volumes of starting culture volume (i.e 50 ml if you grew up 250 mL culture) of a 100 mM solution of LiAc. Mix well, use vortex if necessary.

7. Spin down a final time and resuspend cells in 100 mM LiAc with 15% glycerol to a final volume of 3% of starting culture volume (i.e 7.5 ml if you grew up 250 mL culture).

8. Dispense the cells in 100 µL aliquots in eppendorf tubes and put cells into a cardboard box. Put the carboard box into a styrofoam box and allow to freeze slowly at –80C. The styrofoam box can be removed after the cells are frozen solid (~24h). Unlike E. coli competent cells, quick freezing in liquid nitrogen will severely reduce competency.

These cells can be used according to this protocol.