gray colour extract

Arnoud Kal KAL at amc.uva.nl
Tue Sep 6 11:24:55 EST 1994


Dear readers,
I am trying to break large amounts of yeast cells with glass beads
in a Sorvall Omni-mixer or in a Waring blender. The problem is
that the cell go in as a brownish suspension, but after mixing and
blending the cell extract has a gray colour. I never see this when
I make extracts in eppendorf tubes. Can it be the buffer in combination
with the stainless-steel container or knives? The buffer contains:

200mM Tris pH8
400mM Ammoniunsulfate
10% glycerol
protease inhibitors

The ammoniumsulfate is included because I want to purify a DNA binding
protein, the salt elutes it from the DNA. Can it be left out of the
breaking buffer and added after breaking the cells?
Other suggestions how to break 250-500 grams of cells are very much
appreciated. I used a Dynomill before (it worked) but this was not
in my own lab and at that time I had to break 2kg cells.

Thank you for reading,
Sincerely,

Arnoud Kal, University of Amsterdam, The Netherlands.



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