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more on lacz

Fatima Cvrckova FATIMA at AIMP.UNA.AC.AT
Tue Dec 7 08:32:20 EST 1993


Hello again,
Just one more note concerning lacz assays:
I noticed that people keep mentioning "yeast grown on Xgal plates" and sometimes
also problems with the accuracy of the semiquantitative information coming from
such assays. I am including a protocol for a nice filter betagal assay used 
routinely in our lab. I guess it has been published somewhere, too - look
into some of the old Nasmyth papers on HO. It is fast, simple, cheap (you need
less Xgal) and it provides permanent record (you just need to soak the filter 
in 0.2 M sodium hydrogencarbonate to stop the reaction). And (surprisingly)
you still can rescue live cells from stained filters (fresh ones, maybe not
after the fixation). Good luck!            Fatima

Materials
Liquid nitrogen
Z buffer (60mM-disodium hydrogen phosphate, 40mM-sodium dihydrogen phosphate, 10mM-
KCl, 1mM-magnesium sulphate, 50mM-2-mercaptoethanol)(3.5 5l/ml)

X-gal solution (25mg/ml X-gal in dimethylformamide).

Grow patches or colonies on plates.
Replicate onto nitrocellulose filter (or nylon - works as well).
Carefully submerge in liquid nitrogen (before the filter dries) by placing the 
filter on an aluminium foil boat floating on nitrogen until thoroughly cooled, then 
submerge.

Place filter on Petri dish containing a Whatman No.1 disc saturated with1.8ml Z 
buffer + 255l Xgal solution.
Make sure that there are no bubbles between the filter and disc.

Incubate at 30:C until colour develops(10-60 minutes).

Monitor over time to compare activity of different strains as they might plateau at 
the same "blueness".



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