Beta-gal Assay
互联网
2662
Beta-gal Assay
Adapted from Current Protocols in Molecular Biology
1. Grow 5ml YEPD cultures to mid-log phase.
2. Centrifuge and resuspend cells in 5ml Z-buffer, then place on ice.
3. Measure OD600.
4. Use straight, or dilute cell mix 10x or 20x (40 or 80 brought to 0.8mL with Z-buffer).
5. Using Pasteur pipet, add 1 drop of 0.1% SDS and 2 drops of chloroform to each tube.
6. Vortex well for 15 sec.
7. Equilibrate @ 30 deg C for 15 min.
8. Add 160 of 4mg/ml ONPG, and vortex well for 10 sec.
9. Incubate at 30 deg C and begin timing.
10. Remove after about 15-20 min (empirically determined by color).
11. Quench reaction by adding 400 of 1M Sodium Carbonate.
12. Spin down cell debris.
13. Measure OD420 and OD550.
14. Calculate Units using the following formula:
U= 1000 x [(OD420)-(1.75 x OD550)] / [(Time) x (Vol) x OD600]
Where Vol is volume of culture used in assay in mls, and Time is minutes at 30 deg C.
40mM NaH2PO4
10mM KCl
1mM MgSO4
50mM 2-mercaptoethanol
pH 7.0
DO NOT AUTOCLAVE
To make 0.1M potassium phosphate buffer, pH 7, you first need to make two solutions:
Solution A: 27.2 g KH2PO4 in 1 L water.
Solution B: 34.8 g K2HPO4 in 1 L water.
Mix 39 ml Solution A and 61 ml Solution B and then add 100 ml of water.
2. Centrifuge and resuspend cells in 5ml Z-buffer, then place on ice.
3. Measure OD600.
4. Use straight, or dilute cell mix 10x or 20x (40 or 80 brought to 0.8mL with Z-buffer).
5. Using Pasteur pipet, add 1 drop of 0.1% SDS and 2 drops of chloroform to each tube.
6. Vortex well for 15 sec.
7. Equilibrate @ 30 deg C for 15 min.
8. Add 160 of 4mg/ml ONPG, and vortex well for 10 sec.
9. Incubate at 30 deg C and begin timing.
10. Remove after about 15-20 min (empirically determined by color).
11. Quench reaction by adding 400 of 1M Sodium Carbonate.
12. Spin down cell debris.
13. Measure OD420 and OD550.
14. Calculate Units using the following formula:
U= 1000 x [(OD420)-(1.75 x OD550)] / [(Time) x (Vol) x OD600]
Where Vol is volume of culture used in assay in mls, and Time is minutes at 30 deg C.
Z Buffer:
60mM Na2HPO440mM NaH2PO4
10mM KCl
1mM MgSO4
50mM 2-mercaptoethanol
pH 7.0
DO NOT AUTOCLAVE
ONPG:
4mg/ml in 0.1M potassium phosphate buffer, pH 7, filter sterilized and stored frozen.To make 0.1M potassium phosphate buffer, pH 7, you first need to make two solutions:
Solution A: 27.2 g KH2PO4 in 1 L water.
Solution B: 34.8 g K2HPO4 in 1 L water.
Mix 39 ml Solution A and 61 ml Solution B and then add 100 ml of water.
