Β-Galactosidase Liquid Assay for Yeast

Procedure:

1. Grow the desired yeast strain to log phase in a liquid culture.

2. Measure the Optical Density at 600 nanometers (OD600) by diluting 0.5 ml of culture in 0.5 ml of media (see Hint #1).

3. Place 1 ml of culture into a microcentrifuge tube (see Hint #2).

4. Microcentrifuge at maximum speed for 5 min.

5. Remove the supernatant using a P1000 pipette (do not use a vacuum line).

6. Add 1 ml of Z Buffer and mix well.

7. Microcentrifuge at maximum speed for 5 min.

8. Remove the supernatant using a P1000 pipette (do not use a vacuum line).

9. Resuspend the cell pellet in 150 μl of Z Buffer with 2ME.

10. Add 50 μl of Chloroform (CAUTION! see Hint #4).

11. Add 20 μl of 0.1% SDS.

12. Vortex the sample vigorously for 15 sec.

13. Add 700 μl of pre-warmed (30°C) ONPG Solution.

14. Incubate at 30°C while timing the reaction; remove aliquots at the appropriate time points (see Hint #3).

15. Stop reaction by adding 500 μl of 1 M Na2CO3.

16. Microcentrifuge at maximum speed for 10 min.

17. Determine the absorbance of the supernatant at 420 nm.

18. Calculate as follows:

Miller Units = (420*1000)/(600*min*ml)

Where:

420 is the absorbance units at 420 nanometers

600 is the absorbance units at 600 nanometers

min is the reaction time in minutes

ml is the reaction volume in ml

Solutions

Z Buffer
0.75 g KCl
Bring the final volume to 1 liter using ddH2O
16.1 g Na2HPO4, 7H2O
246 mg MgSO4, 7H2O
5.5 g NaH2PO4

1 M Na2CO3

ONPG
1 mg/ml ONPG (o-Nitrophenyl Β-D-Galactopyranoside)
Prepared in Z Buffer with 2ME

0.1% SDS
0.1% (w/v) SDS

Z Buffer with 2ME
Prepared in Z Buffer
Add 270 μl of 2-Mercaptoethanol per 100 ml of Z Buffer

BioReagents and Chemicals:

Magnesium Sulfate, Heptahydrate
Chloroform
Potassium Chloride
Sodium Carbonate
2-Mercaptoethanol
ONPG
SDS
Sodium Phosphate Monobasic
Sodium Phosphate Dibasic Heptahydrate

Protocol Hints:

1. When reading many samples, use a P200 pipette to remix the cells before reading (since the cells will settle to the bottom of the tube over time). Always determine the optical density of several identical samples and determine the mean optical density value.

2. Do each sample in triplicate, at least.

3. 20 min to 3 hour total reaction time.

4. This substance is a biohazard. Please consult this agent's MSDS for proper handling instructions.

Link: http://www.bio.com/protocolstools/prnprot.jhtml?id=p1203