Standard protocol for preparing yeast cells

Standard protocol for preparing yeast cells for any purpose

pre-culture

Inoculate a yeast strain from plate into ~5 ml of liquid medium (YPD or SD medium) and incubate at least overnight but even better for 2-3 days on a orbital shaker at 200-250 rpm at 30°C. For strain that grow normally, it is enough with just noticeable turbidity

This culture can be inoculated from a plate or directly from a frozen culture in the case of YPD medium. The best containers for this culture are 50 mL round-bottom glass centrifuge tubes with a cotton stopper or aluminium cap. These occupy little space in the incubator and mixing is efficient for 5-10 mL medium.

main culture

A few hours before inoculation, place the sterile medium and the empty glassware (for example, a 250 ml Erlenmeyer flask at 30°C. This way the medium is preheated to the correct temperature and this will minimize the lag phase.

Determine the titer of the yeast culture by pipetting 50 µL of cells into 1.0 ml of water in a cuvette and measuring the Optical density (OD) at 640 nm using a spectrophotometer. You can use pure water as standard, the OD increase due to the medium is negligible.

$$X=\frac{\text{culture volume}\times OD640_{\text{start}}}{\frac{1050}{50}\times OD640_{\text{pre-culture}}-OD640_{\text{start}}}$$

Variable	Meaning
$X$	Volume of pre-culture to add (mL)
$OD640_{\text{pre-culture}}$	Optical density of the pre-culture at 640 nm as measured by instrument
$OD640_{\text{start}}$	Desired starting optical density
$\text{culture volume}$	Initial volume of the culture medium
$\frac{1050}{50}$	Dilution or scaling factor used in the calculation

About 1-2 mL of a saturated S. cerevisiae CEN.PK culture added to 50 mL of medium gives an OD640 = 0.17 as measured using a GENESYS20 spectrophotometer which corresponds to 5 * 10⁶ cells/mL.

Incubate the culture until the desired optical density. For transformation, 2-4 generations are recommended.