Sonics VC750 - Manual (Page 27)

The ability to control the amplitude at the probe tip is a prerequisite for process
optimization. And because each application requires its own set of processing
parameters, due to variation in volume and composition, the optimum amplitude can
only be determined empirically. When processing a new sample, it is recommended
that the amplitude be set first at 50% (30% with a microtip) and then increased of
decreased as required.
Prior to sonication, cells can be treated with various agents to aid the disruption
process. Lysis can be promoted by suspending cells in a hypotonic buffer, which
causes them to swell and burst more readily by physical shearing. Lysozyme can be
used to digest the polysaccharide component of yeast and bacterial cell walls.
Alternatively, processing can be expedited by treating tough cells with glass beads to
facilitate the crushing of cell walls. This treatment is commonly used with yeast cells.
Viscosity of a sample typically increases during lysis due to the release of nucleic acid
material. DNase can be added to samples (25-50 µg/ml) to reduce this problem.
Nuclease treatment is not required for sonicated material because sonication shears
chromosomes. Because proteolysis can be a problem whenever cells are
manipulated; it is advisable to add protease inhibitors to samples undergoing lysis.
NOTE:
All living organisms contain proteolytic enzymes (proteases and peptidases).
Proteases are required for a variety of cellular functions, such as cellular repair or the
digestion of extracellular material. In whole cells, protease activity is tightly regulated
by compartmentalization or inhibitors to prevent damage to cellular proteins. Cell lysis
disturbs this regulation and proteolytic degradation of the sample becomes a
concern. Therefore, addition of protease inhibitors to cell lysis buffers is often
required. Protease inhibitors are biological or chemical compounds that functions by
reversibly or irreversibly binding to the protease. Proteases generally belong to one of
four evolutionarily distinct enzyme families based on the functional groups involved in
cleavage of the peptide bond. Therefore, several different types of inhibitors are
generally required to protect proteins from proteolysis during extraction and
purification.
Cellular disruption is the first step in RNA isolation and one of the most critical steps
affecting yield and quality of the isolated RNA. Typically, cell disruption needs to be
fast and thorough. Slow disruption, for example placing cells or tissue in guanidinium
isothiocyanate (GITC) lysis solution without any additional physical shearing, may
result in RNA degradation by endogenous RNase released internally, yet still
inaccessible to the protein denaturant, GITC. This is especially a concern when
working with tissues high in endogenous RNase such as spleen and pancreas.
Incomplete disruption may also result in decreased yield because some of the RNA in
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