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A Dohlman Lab Protocol

1. Start with a plate of yeast colonies to be tested (often the result of the transformation of a library or a round of mutagenesis).

  • These colonies should contain the lacZ gene under the control of the FUS1 promoter.

2. For each plate to be tested, place a circle of 3MM Whatman paper into each of two empty petri dishes.

  • The Whatman paper should cover the entire bottom of the petri dishes.

3. Add 2.5 ml of a-factor to the Whatman paper at the bottom of one of the petri dishes.

  • Use a concentration of a-factor that produces a different response in wild-type versus mutant strains. This must be determined empirically.
  • If there are any bubbles, smooth them out with forceps.

4. Label a circular piece of nitrocellulose (NC) filter the size of the petri dish (GelmanSciences, BioTrace® NT 82mm, #66487) with a ball point pen.

5. Pick up the NC filter with tweezers and place it carefully across the plate of yeast colonies so that there are no bubbles or wrinkles in the filter. Let sit for 1-2 min.

6. While the NC filter is sitting on the yeast colonies, dip a needle in india ink and poke holes through the filter and into the solid media. This enables positive colonies on the filter to be matched with the colonies on the plate from which they were lifted.

7. Transfer the NC filter to a petri dish containing a-factor, colony side up.

8. Cover the dish and seal with parafilm. Put at 30° for 2.5 hrs.

9. During the 2.5 hr incubation, aliquot 2.5 ml of Z buffer + X-gal into the remaining petri dishes containing Whatman paper.

Z buffer + X-gal:
60 mM Na2HPO4
40 mM NaH2PO4oH2O
10 mM KCl
1 mM MgSO4o7H2O
39 mM 2-mercaptoethanol
1 mg/ml X-gal
Adjust pH to 7.0.

  • Z buffer without X-gal and without 2-mercaptoethanol can be stored at room temperature.
  • X-gal should be stored as a 100 mg/ml stock in dimethylformamide (DMF) at -20°C.

10. Remove the plates from 30°, lift the NC filter off the Whatman paper, and place on an aluminum foil “boat.” Then, slowly lower into an ice bucket containing liquid nitrogen.

  • Freezing the cells in this way serves to permeabilize the membrane and allow the subsequent entry of X-gal, the substrate for b-galactosidase.
  • The aluminum foil boat avoids having to maneuver the NC filter with tweezers. (The NC gets very brittle when frozen.)

11. After about 10 sec, carefully raise the boat to remove the NC filter from the liquid nitrogen.

12. Allow the NC filter to warm to room temperature (about 1 min).

13. Place the NC filter, colony side up, in the petri dish containing Z Buffer + X-gal.

14. Cover the dish and seal with parafilm. Put at 30° overnight.

  • Blue color may start to appear after a few hours.