Herskowitz Lab Protocol
From : RKT 7/96

Bgal Filter Assays

  1. Grow the desire yeast on any appropriate plate
  2. Obtain the plate of the type required for the assay (eg. YEP-galactose or YEP-D, etc)
  3. Place a Whatman 50 circular filter on the plate without bubbles using clean forceps
  4. Replica the growing yeast onto the filter
  5. incubate plate until yeast are substantially visible colonies or patches
  6. prepare 3ml Z buffer per plate, add 20ul of 2% X-gal in DMF per plate [store at -20], and 8ul of b-mercapto-ethanol per plate
  7. aliquot 3ml of the above mix into an empty plastic petri dish
  8. place a Whatman #3 filter in the petri dish
  9. using forceps remove the filter containing the yeast
  10. dip the filter into liquid nitrogen for 5-10 seconds
  11. remove and thaw for 20s-60s
  12. overlay the filter (yeast up) onto the #3 filter in the plastic petri plates, avoid any bubbles between the two filters
  13. incubate at 37 deg until the color substrate is visible on the positive control.

Z buffer is
Na2HPO4.7H2O 16.1g (8.5g anhydrous)
NaH2PO4.H2O 5.5g
KCL 0.75g
MgSO4.7H2O 0. 246g ---> 1L

Related protocols include a quantitative liquid assay and a semi-quantitative agarose overlay assay.

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