"Yeast lytic enzyme" versus Zymolase
mrclean at u.washington.edu
Fri Jan 28 02:48:56 EST 1994
I have been trying to utilize the protocol below to make crude whole
yeast cell extracts from single S. cerevisiae colonies in order to screen
mutants for topoisomerase activity. As you will see, the protocol calls
for the use of Zymolase (ICN). My advisor felt that Zymolase was
prohibitively expensive to use when screening large numbers of mutants
(especially on the long shot experiment that we're trying to perform).
He asked me to try an alternative enzyme described in the ICN catalog as
"Yeast Lyric Enzyme-70,000." I ordered this enzyme and attempted to use
it as a substitute for Zymolase in the protocol by using a comparable
number of lytic units. Unfortunately, my first attempts using this
enzyme as a substitute have failed. I am now considering increasing the
amount of enzyme that I add during the spheroplast step. Has anyone had
experience using "yeast lytic enzyme" to generate S. cerevisiae
extracts? Do you have any suggestions as to how much of the enzyme
should be used in this situation? Should I even be trying to use "yeast
lytic enzyme" rather than Zymolase?
Any advice or information that you provide would be greatly appreciated.
Department of Microbiology
University of Washington
1. Scrape a single colony from a fresh plate.
2. Resuspend the colony in 30 microliters of SED buffer (1M sorbitol,
25 mM EDTA, 50 mM DTT). 3. Incubate at 37 degrees C for 30 min.
4. Add 4 microliters of Zymolase 60000 (2.5 mg/ml). [I found that
Zymolase 60,000 is no longer available from ICN. ICN now sells Zymolase
100T (100,000 U/gm) and Zymolase 20T (20,000 U/gm).]
5 Incubate at 30 degrees C for 30 min.
6. Pellet spheroplast in microfuge and resuspend pellet in 12
microliters of yeast lysis buffer (20 mM TRIS-HCl, pH 7.5; 1 mM EDTA; 1
mM DTT; 1 mM PMSF; 500 mM KCl; 10% glycerol).
7. Incubate on ice for 30 min.
8. Centrifuge 10 min. Assay 1 microliter of supernatant in a standard
10 microliter topoisomerase assay.
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