Yeast - methods

LiAc Yeast Transformation

Solutions:

Synthetic drop out solution 10 x in AD: L-isoleucine 300 mg/L, L-valine 1.5 g/L, L-adenine hemisulfate salt 200 mg/L, L-arginine HCl 200 mg/L, L-histidin HCl monohydrate 200 mg/L, L-leucine 1 g/L, L-lysine HCl 300 mg/L, L-methionine 200 mg/L, L-phenylalanine 500 mg/L, L-threonine 2 g/L, L-tryptophan 200, L-tyrosine 300 mg/L, L-uracil 200 mg/L

SD -Trp medium (synthetic dropout medium): synthetic minimal medium lacking tryptophan: yeast nitrogen base without amino acids 6.7 g/L,  2 % dextrose (glucose) (sterile dextrose solution is added after autoclaving to avoid maillard reactions), pH adjusted to 5.8, for plates : agar 1.5 g/L

YPD (yeast peptone dextrose) broth, yeast complete medium: yeast extract 10 g/L, peptone 20 g/L, 2 % dextrose (glucose), pH adjusted to 5.8

Aqua dest. sterile

LiAc 100 mM sterile

LiAc 1 M sterile

Bacterial RNA, used as carrier

Poly-ethyleneglycol PEG 50 % (w/v) sterile filtered

10 mL of SD -Trp medium are inoculated with the appropriate yeast strain and incubated at 30°C while shaking at 200 rpm o/n. On the next day OD at 600 nm is measured and the yeast culture is diluted with YPD to OD₆₀₀ 0.1. A total volume of 50 mL diluted yeast culture is used for further incubation. Every hour OD₆₀₀ is measured until OD₆₀₀ 0.4 is reached (3 - 5 hours). Then the cell number is calculated with a Thoma chamber. 2x10⁷ cells/mL are sufficient for 10 transformations. The yeast is then harvested by centrifugation at 3000 rpm for 5 minutes, the supernatant is carefully removed and collected for autoclaving. The pellet is resuspended in 25 mL sterile AD and again centrifuged at 3000 rpm for 5 minutes. After removing of the supernatant the pellet is resuspended in 1 mL LiAc 100 mM. Excess of LiAc is removed by spinning the tubes for 15 seconds at full speed in a tabletop centrifuge and carefully removing the supernatant. The yeast pellet is brought to a final volume of 500 µL with LiAc 100 mM. Aliquots of 50 µL are prepared. One 50 µL aliquot of this yeast suspension is used for one transformation. 50 µL aliquots are again briefly centrifuged to pellet the cells, the supernatant is removed and on top of the yeast pellet, layers of the following transformation solutions are pipetted in following order: 240 µL 50 % PEG, 36 µL LiAc 1 M, 3.3 µL of bacterial RNA (31 µg/µL), 70.7 µL sterile AD, 1 µg plasmid DNA (1µg/µL). The tube is then thoroughly mixed by vortexing for 1 minute until the yeast pellet is completely dissolved and placed for 30 minutes in a 30°C water bath. The tube is then transferred to a 42°C water bath for 25 minutes in order to perform the heatshock. The transformation mix is then briefly centrifuged for 15 seconds at 4 000 x g (7 000 rpm) in a table top centrifuge, the supernatant is discarded and the pellet is resuspended in 1 mL sterile AD. 50 µL of this transformed yeast suspension are plated on SD – Leu, - Trp, - Ade plates and incubated at 30°C for some days.