Could anyone please tell me how to prepare cell free extract for enzyme for gram positive bacteria skipping sonication step?
we do not sonification but french press for cell breakage during CF extract preperation
Dear Sneha Gupta, I mean that only French pressure (ultra sonication)
Dear Pooja,
You can try treating your cells with lysozyme. This will weaken/remove the cell wall. Then, breakage will be easier. Another way is to use a blender with very tiny glass beads added to the blender cup. When the blender is turned on, the beads + cells are suspended simultaneously. When the beads strike the cells, they will be broken. A blender with a stainless steel blender cup is recommended, but glass is OK, too. It will help if you can get blender cups with a polytron head (that is, the blade assembly). This is a little different from the normal blade assembly in a household blender, and provided much better shearing action and will do a better job breaking the cells.
The glass beads will make a big difference. I've used a blender w/ glass beads to make cell-free extracts of yeast, which are very hard to break, without any pre-treatment.
If you decide to try this, do it in a cold room, as the blender cup will get warm while it is running.
I hope this helps you.
Bill Colonna, Center for Crops Utilization Research, Iowa State University, Ames, Iowa, USA. [email protected]
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