pH

Fatima Cvrckova FATIMA at AIMP.UNA.AC.AT
Sat Jan 15 04:46:50 EST 1994


Dear buddies and pombies,
(I don't know that much about pombe but I suspect that it acidifies media, too),

just two notes into the ongoing discussion on pH:

1. The secret of keeping pH of a yeast culture high ist simple: buffer the medium
with something the yeast does not eat. Phosphate is being taken up quite rapidly
and efficiently. I don't have any experience of my own but I dimly recall that 
people have been using succinate.

2. To assay lacZ activity in yeast, you don't have to grow them on Xgal at pH 7
(they don't particularly like pH 7, anyway). I am appending the famous Nasmyth
protocol for Xgal assays on filters, which has at least two advantages - you 
save Xgal and therefore money and you can keep a permanent record.

Good luck!

Fatima

Protocol follows:

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

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

Grow patches or colonies on plates.
Replicate onto nitrocellulose filter and grow o/n.
Carefully submerge the filter in liquid nitrogen (before the filter dries) by
placing the filter on an aluminium foil boat floating on nitrogen until 
horoughly cooled, then submerge.

Place filter on Petri dish containing a Whatman No.1 disc saturated with
1.8ml Z buffer + 25µl Xgal solution.
Make sure that there are no bubbles between the filter and disc.

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

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

You can pick live cells directly from the filters (a few percent still 
survive).

If you soak the filter in 0.2 M NaHCO3, you can dry it and keep it 
indefinitely for record.



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