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β-gal assays

Filter based assay

  1. Lay a nitrocellulose filter (Nylon may work too) on a YEPD plate.
  2. Replica plate the yeast directly onto the filter and grow O/N.
  3. Prepare an empty petri dish with a Whatman filter paper.
  4. Add to this 6 ml of the X-Gal mixture in Z-buffer.
  5. Dip the nitrocellulose filter in liquid N2 for 30 sec. and thaw at RT.
  6. Place the filter on top of the Whatman paper, colonies up.
  7. Put at 37°C up to 24 hrs, score also at earlier points.

X-Gal mixture
100 µl 4% X-Gal in DMF N,N-dimethyl formamide
6 ml Z-buffer
11µl β-mercaptoethanol

Z-buffer (1L)

Na2HPO4·7H2O 8.52 g (or 6.1 g anhydrous)
NaH2PO4·H2O 5.5 g (or 4.8 g anhydrous)
KCl 0.75 g
MgSO4·7H2O 0.246 g (or 0.12 g anhydrous)

pH should be 7.0

Agar overlay assay

Adapted from Xiaofeng Xin, Boone Lab, University of Toronto

  1. Grow yeast on YEPD at 30°C O/N, or on -Leu -Trp at 30°C for 2days. The latter is recommended because of its clear background.
  2. Determine the gel volume needed.

    Plate type Volume for one plate
    Petri dish (10 cm) 10
    Petri dish (15 cm) 30
    Omnitray 20
  3. Weight low melting agarose into a big flask (enough room for boiling), add KPO4 buffer, microwave at high power in interval until the gel is clear. Then add SDS and DMF. If the gel is not homogeneous, microwave a little bit more. Use gloves to handle DMF, as it it toxic (plus it penetrates everything, including many types of gloves).

    Gel volume (ml) 20 40 60 80 100
    Low melting agarose (g) 0.1 0.2 0.3 0.4 0.5
    0.5 M KPO4 (ml) 18.6 37.2 55.8 74.4 93
    10% SDS (ml) 0.2 0.4 0.6 0.8 1.0
    DMF (ml) 1.1 2.2 3.3 4.4 5.5
  4. Cool the gel to 55°C, add X-Gal stock, mix well and overlay onto the yeast plates using pipette.

    Gel volume (ml) 20 40 60 80 100
    4% X-gal in DMF (ml) 0.1 0.2 0.3 0.4 0.5
  5. Keeping the plates in the dark, let agar solidify at room temperature, then move to 30°C. Score when negative controls are still white, and controls turned blue (5-8 hours).

0.5 M KPO4 buffer pH7.0 (1L)

1M KH2PO4 195 ml
1M K2HPO4 305 ml
H2O 500 ml