Herskowitz
Lab Protocol
From : RKT 7/96
Bgal Filter Assays
- Grow the desire yeast on any appropriate plate
- Obtain the plate of the type required for the assay (eg. YEP-galactose or YEP-D, etc)
- Place a Whatman 50 circular filter on the plate without bubbles using clean forceps
- Replica the growing yeast onto the filter
- incubate plate until yeast are substantially visible colonies or patches
- 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
- aliquot 3ml of the above mix into an empty plastic petri dish
- place a Whatman #3 filter in the petri dish
- using forceps remove the filter containing the yeast
- dip the filter into liquid nitrogen for 5-10 seconds
- remove and thaw for 20s-60s
- overlay the filter (yeast up) onto the #3 filter in the plastic petri plates, avoid any bubbles between the two filters
- 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.
