Standard protocol for preparing yeast cells - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

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. 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. These occupy little space in the incubator and mixing is efficient for 5-10 mL medium.

Place a bottle of medium and a 250 ml culture flask in the same or another 30°C incubator as well. 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 spectrophotometer cuvette and measuring the OD at 640 nm. You can use pure water as standard, the OD increase due to the medium is negligible.

OD640 * 1050 / 50 * Inoculation(mL) = Culture_volume(mL) * OD640(init)

Add 50 mL of the pre-warmed YPD to the pre-warmed culture flask then add 2.5 x 10e8 cells to give 5 x 10e6 cells/ml. About 1-2 mL of a saturated CEN.PK culture gives an OD640 = 0.17 using a spectrophotometer GENESYS20 which corresponds to 5 * 10⁶6 cells/mL.