Beta-Galactosidase Assay

  1. Grow 5ml YPD cultures to mid-log phase.

  2. Centrifuge and resuspend cells in 5ml Z-buffer, then place on ice.

  3. Measure O.D.(600).

  4. Use straight, or dilute cell mix 10x or 20x (40 or 80ul 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 seconds.

  7. Equilibrate at 30oC for 15 minutes.

  8. Add 160ul of 4mg/ml ONPG, and vortex well for 10 sec.

  9. Incubate at 30oC and begin timing.

  10. Remove after about 15-20 minutes (empirically determined by color).

  11. Quench reaction by adding 400 ul of 1M sodium carbonate.

  12. Spin down cell debris.

  13. Measure O.D.(420) and O.D.(550).

  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 30oC.


Z BUFFER
(60mM Na2HPO4, 40mM NaH2PO4, 10mM KCl, 1mM MgSO4, 50mM 2-mercaptoethanol, pH 7.0)

TO MAKE 100ml: 

  • 6.0ml 1M Na2HPO4 stock solution
  • 4.0 ml NaH2PO4 stock solution
  • 0.33 ml 3M KCL stock solution
  • 1.0ml 100mM MgSO4 stock solution
  • 350 ul 2-mercaptoethanol
Bring volume to 100 ml with distilled H2O. Do not autoclave!

ONPG

To make 10ml: 

  • 40 mg ONPG
  • 10.0 ml 0.1 potassum phosphate buffer pH7.0


0.1M potassium phosphate buffer

To make 100ml: 

  • Make solution A: 27.2 g KH2PO4 in 1 L water.
  • Make 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.