Yeast Cell Competent Recombination Protocol

Yeast cells competent for recombination were cultured in YPR medium at 30°C to an OD600 of 1.0. Cells were then simultaneously arrested in G1 phase by addition of alpha factor (50 ng/mL) and expression of R recombinase induced by addition of galactose to a final concentration of 2% (w/v). Cells were grown for an additional 2h at 30°C before harvesting by centrifugation (10min, 7.000 g at 4°C), yielding approximately 1.5g of yeast cells wet weight per liter of medium. Cells were resuspended with water, before being pelleted in sealed 25mL syringes by centrifugation (10min, 7.000 g at 4°C). The supernatant was decanted, the syringe unsealed and the cells were extruded into liquid nitrogen. The resulting cell “spaghetti” can be stored at −80°C until further usage.