All liquid cultures are performed in 14ml Falcon tubes, with caps loose (not tightly clipped), at 30°C, 220 rpm.

Book the the cytometer for day 4, from 3 to 5 p.m.


Day 1 : Streak relevant strain on YPD plates for single colonny isolation.

Day 3 : Inoculate 3ml of YPD with a single colonny

Day 4 :

1) Measure OD600nm of 1:10 culture dilutions in YPD. Prepare a series of tubes containing 4ml of SD-Met1mM. Process only 3 to 4 tubes at a time. Apply a 10 minute delay between each pair of tubes. Inoculate at the appropriate time with the equivalent of 0.1 OD.
Culture for 3 hours at 30°C.
    
    Prepare SC-Met50μM.

2) After 3 hours of incubation. Spin 5 min at 4000 rpm  in Beckman. Discard supernatant. Resuspend cells in 4ml of SC-Met50μM .
Culture for 2 hours at 30°C.

3) Prepare cytometer. Just before measurement, put 20μl of culture in 1ml TBS, and analyse (keep flow < 200 cells/s).

Culture Medium:

MET Stock 0.1M: Dissolve methionine at 0.1M final in water, filter-sterilize and store at 4°C.

Mix dropout-Met: all desired amino-acids, except methionine, as a powder (AD 1g, UR 2g, A 2g, R 2g, D 2g, N 2g, C 2g, E 2g, Q 2g, G 2g, H 2g, I 2g, L 4g, K 2g, F 2g, P 2g, S 2g, T 2g, W 2g, Y 2g, V 2g).

SC-M medium:             for 1 liter
Yeast Nitrogen Base with Ammonium sulfate    6,7g
Glucose    20g
Mix dropout -Met    2g
Water    To 1 liter
NaOH 1M    1ml
Autoclave at 0.5 bar.

SC-Met1mM: SC-M medium plus MET Stock 0.1M to reach 1mM final
SC-Met50μM: SC-M medium plus MET Stock 0.1M to reach 50μM final