Yeast Galactose Induction

Day 0: Buffer Making

20% Raffinose – Do not autoclave. Filter-sterilize. 

Raffinose	20 g
MPW	100 mL

*Solubility of Raffinose: 203 g/L (25 °C)

20% Galactose – Do not autoclave. Filter-sterilize. 

Galactose	20 g
MPW	100 mL

* Solubility of Galactose: 650 g/L (20 °C)

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

2X SC-Ura-Gluc	25 mL
20% Raffinose	5 mL
20% Galactose	5 mL
MPW	15 mL
Total Volume	50 mL

1x SC+Ura+2% Raffinose+2% Galactose+1X Uracil (1st O/N Media: Neg Ctrl)

2X SC-Ura-Gluc	25 mL
20% Raffinose	5 mL
20% Galactose	5 mL
10X Uracil	5 mL
MPW	10 mL
Total Volume	50 mL

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

2X SC-Ura-Gluc	25 mL
20% Galactose	5 mL
MPW	20 mL
Total Volume	50 mL

1x SC+Ura+2% Galactose (2nd O/N Media: Neg Ctrl)

2X SC-Ura-Gluc	25 mL
20% Raffinose	5 mL
20% Galactose	5 mL
MPW	15 mL
Total Volume	50 mL

 

Day 1:

1. Make O/N cultures of the Neg control (Untransformed BY4741) in 1X SC+Ura + 2% Raffinose + 2% Galactose
2. Make O/N cultures of the constructs in 1X SC-Ura + 2% Raffinose + 2% Galactose.
3. Incubate overnight at 30 ˚C and rpm 220.

Day 2:

1. Make O/N cultures of the Neg control (Untransformed BY4741) in 1X SC+Ura + 2% Galactose.
2. Make O/N cultures in 1X SC-Ura + 2% Galactose.
3. Incubate overnight at 30 ˚C and rpm 220.

Day 3:

1. Set up the Plate Reader Assay.
2. Neg control: in 1X SC+Ura + 2% Galactose
3. Constructs: in 1X SC-Ura + 2% Galactose

[Making Frozen Stocks]

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.