Yeast Galactose Induction

Day 0: Buffer Making

20% Raffinose

**ALWAYS Store at 4˚C / Do not store more than 1 month

	mol	FW
Raffinose			20 g
MPW			100 mL

1. Autoclave 25 min

20% Galactose

**ALWAYS Store at 4˚C / Do not store more than 1 month

	mol	FW
Galactose			20 g
MPW			100 mL

1. Autoclave 25 min

1x SC-Ura+2% Raffinose+2% Galactose (O/N Media)

2X SC-Ura	10 mL
20% Raffinose	0.2 mL
20% Galactose	0.2 mL
MPW	9.6 mL

1x SC-Ura+2% Galactose (Day Culture Media)

2X SC-Ura	5 mL
20% Galactose	2 mL
MPW	3 mL
Total Volume	10 mL

1. Make four O/N cultures in 1X SC-Ura + 2% Raffinose + 2% Galactose as dilution series. For the first tube, pick a yeast colony from the plate and resuspend it by pipetting. Dilute the subsequent tubes by 1/10.
2. Incubate at 30 ˚C, overnight

 

Day 1:

1. Measure OD600 of the O/N culture.
2. Pick the O/N culture at the log phase (OD between 0.4~0.6)
3. Spin down the chosen culture at 4˚C, for 5 mins, at 3000 rpm (=845 rcf)
4. Decant the supernatant and wash the pellet x3 with MPW: 4˚C, for 5 mins, at 3000 rpm (=845 rcf)
5. After washing, add 5 mL of MPW and resuspend by pipetting
6. Prepare microfuge tubes according to the number of different day culture media that you will test on the Plate Reader.
7. Transfer 1 mL of the resuspension to each microfuge tube.
8. Spin down the microfuge tubes to remove MPW:  4˚C, for 3 mins, at 14,000 rpm (=18,407 rcf)
9. Carefully decant the MPW.
10. Dispense corresponding day culture media to the microfuge tubes to prepare yeast day cultures. At this point, the prepared yeast day culture will have the same OD as the starting O/N culture.
11. Set up the Plate Reader Assay at 30 ˚C in 1X SC-Ura+2% galactose day culture media.