1. Replicate plate onto Nitrocellulose filter (NC filter). 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.

2. Mark the position of the filter by dipping a needle in India ink and poking holes through the filter and into the solid media.

Cells on the NC-filter can be treated (e.g., a-factor) by placing it (colony side up) in a Petri dish containing the desired reagent.

3. Use tweezers to lift filter and slowly submerge in liquid Nitrogen for 1 minute. Caution: the NC-filter is very brittle when frozen.

4. Thaw at the room temperature.

5. Add 2 ml Z-buffer (40 mM Na₂HPO₄, 60 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄) plus 10 mM DTT to a fresh petri dish. Add 100 µl X-GAL solution (20 mg/ml).

6. Place a Whatman filter disk (9 cm) to absorb buffer.

7. Place the NC-filter on top of the Whatman filter, avoid bubbles.

8. Incubate at 30 °C 30 minutes to overnight.