Please find the following protocol:

Quantitative β-Galactosidase Assay for Meiotic Genes in Yeast

Overview:

This assay is used to measure the gene expression of meiotically-expressed genes during meiosis using the lacZ reporter gene. The lacZ gene is cloned behind a meiosis-specific yeast promoter.


Procedure:


Growth of Cells for Meiotic Time Course

1. Grow yeast SK1 strains to saturation in 2.5 ml YEPAD Medium.

2. Dilute a saturated culture 1:100 in 100 ml YPA Medium and grow at 30°C for 12 to 14 hours (to between 2 x 107 to 5 x 107 cells/ml).

3. Centrifuge the cells in a table-top centrifuge at 2,000 X g for 5 min to pellet cells and discard the media.

4. Resuspend the cells in 300 ml of 2% Potassium Acetate.

5. Remove two 10 ml aliquots for the 0 hour Time Point.

6. Centrifuge the zero (0) hour Time Point cells in a table-top centrifuge at 2,000 X g for 5 min to pellet cells and discard the supernatant. Store cells at -70°C for the β-Galactosidase assay.

7. Incubate the sporulating culture at 30°C with shaking.

8. Remove aliquots at 1.5 to 2 hour intervals: take duplicate 10 ml aliquots for β-Galactosidase assay and a 500 μl aliquot for DAPI Analysis.

9. To the sample for DAPI Analysis immediately add 50 μl of 37% Formaldehyde, mix well, and keep at 4°C.

10. Centrifuge the 10 ml β-Galactosidase Assay aliquots in a table-top centrifuge at 2,000 X g for 5 min to pellet cells



β-Galactosidase Assay

1. Resuspend cell pellets in 500 μl Z Buffer (this should be approximately 5 x 107 cells).

2. Add 5 μl of Zymolyase Solution and incubate at 30°C until spheroplast formation is 95% complete. Check under a microscope for spheroplast formation. Spheroplasts are not as refractive as intact yeast cells. They often look "dark" in the field.

3. Add 6 μl of 2.5% SDS and 15 μl of 50 mM PMSF.

4. Vortex vigorously for 10 sec and incubate at 28°C for 10 min.

5. Vortex for 1 to 2 minutes. Samples can be stored at 4°C at this step.

6. Prepare assays in 2 ml microcentrifuge tubes:

Per assay add:

-> 0.2 ml ONPG solution

-> (X) ml extract from Step #6
(usually between 0.01 to 0.2 ml)

-> Z buffer to a final volume of 1.2 ml

-> BEGIN TIMING when the extract is added to the
assay tube. Add extract last.

7. Incubate at 28°C for 15 min to 20 hr. Stop the reaction when the samples reach a pale yellow color.

8. Stop reaction by adding 500 μl of 1 M Sodium Carbonate and NOTE THE TIME.

9. Centrifuge the microcentrifuge tubes for 3 min in a microcentrifuge to pellet any cellular debris.

10. Decant the supernatant and measure the absorbance of the supernatant at 420 nanometers.

11. Determine protein concentration on a 5 μl aliquot of cell extract (see appropriate protocol for Protein Quantification such as Bradford Assay).

12. Calculation of β-Galactosidase activity:

Activity = nM ONPG cleaved/min/mg yeast protein = [(Abs420) X 1.7)] / [(0.0045) x (time) x (mg/ml protein in extract) x (ml extract used)]

Where x is multiplication

Abs420 is the absorbance of the supernatant at 420
nanometers

Time is time in min.



DAPI Analysis

1. Deposit 100 μl of fixed cells on poly-L-lysine coated slides and remove excess.

2. Add 5 to 10 μl of DAPI Mountant and cover with a coverslip.

3. View slides in fluorescence microscope to assess meiotic stage.


Solutions:

YPA Medium
10 g/liter Yeast Extract
Autoclave
20 g/liter Peptone
30 g/liter Potassium Acetate
Adjust pH to 5.8 with Glacial Acetic Acid

DAPI Mountant
70% (v/v) Glycerol
2% (v/v) n-Propyl Gallate
Prepare in PBS
1 μg/ml 4,6-Diamidino-2-phenylindole (DAPI)

1 M Sodium Carbonate
Autoclave and store at room temperature

ONPG Solution
Dissolved in ddH2O and stored at -20°C
4 mg/ml O-Nitrophenyl-beta-D-Galactopyranoside

50 mM Phenylmethylsulfonyl Fluoride (PMSF)
Prepared in DMSO (CAUTION Biohazard!)
50 mM PMSF

PBS
4.3 mM Sodium Phosphate, Dibasic (Na2HPO4)
pH 7.2
2.7 mM KCl
1.8 mM Potassium Phosphate, Monobasic (KH2PO4)
137 mM NaCl

2.5% SDS
2.5% (w/v) SDS

Zymolyase Solution
50 mg/ml Zymolyase
0.1 M Potassium Phosphate, Monobasic (NaH2PO4), pH 7.5
2 μl/ml of 2-Mercaptoethanol
14 M 2-Mercaptoethanol

YEPAD Medium
10 g/liter Yeast Extract
Autoclave
20 g/liter Glucose
20 g/liter Peptone

Z Buffer
Add 0.66 ml of 14 M 2-Mercaptoethanol
0.19 g KCl
0.06 g Magnesium Sulfate, septahydrate
Autoclave
2.09 g Sodium Phosphate, Dibasic, Anhydrous
(Na2HPO4-H2O)
Adjust pH to 7.0 using NaOH or Phosphoric Acid
1.38 g Sodium Phosphate, Monobasic, Anhydrous
(NaH2PO4-H2O)
Prepare in 250 ml of ddH2O

2% Potassium Acetate
2% (w/v) Potassium Acetate



Bioreagents and Chemicals:

Formaldehyde
Zymolyase
Potassium Phosphate, Monobasic
Glycerol
n-Propyl Gallate
Sodium Hydroxide
Yeast Extract
Peptone
Potassium Acetate
Potassium Phosphate, Dibasic
Phosphoric Acid
2-Mercaptoethanol
Potassium Chloride
Sodium Chloride
Sodium Phosphate, Monobasic, Anhydrous
Sodium Carbonate
Sodium Phosphate, Dibasic, Anhydrous
O-nitrophenyl-beta-D-galactopyranoside
SDS
Magnesium Sulfate, 7-Hydrate
4,6-Diamidino-2-phenylindole
Glacial Acetic Acid
Phenylmethylsulfonyl Fluoride
Glucose


Reference Link:

http://www.bio.com/protocolstools/protocol.jhtml?id=p209