Team:Aachen/Wetlab/Protocol38

Yeast gal assay

  1. Prepare a overnight culture in 5 ml medium with Galactose carbon source (e.g. YPG) and grow at 30°C.

  2. Dilute to OD 0.6 and let the cells grow to OD 0.8-1.
  3. Centrifuge the cells at 4000g.
  4. Discard the supernatant. Resuspend the cells in 1 ml of Z buffer.

  5. Add 3 drops of chloroform and 2 drops of 0.1% SDS. Vortex at top speed for 10 seconds.
  6. Preincubate the samples at 28°C for 5 minutes. Start the reaction by adding 0.2 ml of ONPG.
  7. Stop the reaction by adding 0.5 ml of Na2CO3 stock solution when the sample in the tube has developed on a pale yellow color. Note the ammount of time elapsed during the assay.
  8. Remove the cell debris by centrifuging for 10 minutes in a microfuge and then disscard the pellet.
  9. Measure the OD420 of the reactions.
  10. Express the activity as beta-galactosidase units:
    OD420/(OD600 of assayed culture x volume assayed x time)