UNIVERSITY OF ELLINOIS

DEPARTMENT OF BACTERIOLOGY

362 NOYES LABORATORY OF CHEMISTRY
URBANA

August 16, 1956

Professor J. Lederberg
Department of Genetics
University of Wisconsin
Madison, Wisconsin

Dear Josh:

Jere is a description of the shocking procedure which
I have been promising to send on to you. ile make the protoplasts
the same way that you do. They are then spun down and subjected
to one of two procedures: (a) The pellet is stirred to make sub-
sequent resuspension easier. A volume of distilled water equiva-
lent to 1/8 of the original volume is added to the pellet. A cork
is inserted in the tube and shaken vigorously for uniform suspension.
One then adds concentrated sucrose to yield a final concentration
of 10%. The material is then spun and the pellet collected. (b)
The protoplasts are spun and resuspended in 18% sucrose (or 0.5
Na suéhate) 185 casein hydrolysate, 0.6% HDP, 0.1M K Cl, 107SM
Mg ci, 10-°M Mn Cly. The volume used should yield an eight-fold
concentration of the protoplasm. Ten volumes of distilled water
are added suddenly and within a minute concentrated sucrose added
to yield a final concentration of 10%, The material is spun and
the pellet collected. The initial suspending medium is also an
induction medium when further supplemented with 1000 units of
penicillin and 5 x 1074 TMG, The medium should have a pH of 6.9.
You will note that we can use either sucrose or succinate. .
This has turned out to be true both for shocking and enzyme induction.
For the synthesis of RNA, succinate seems to be superior in the coli
shockates, but this is really still under investigation.

Sincerely yours,

Sok

Se Spiegelman
Professor of Bacteriology

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