Sonics VCX750 - Manual (Page 28)

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shown to effectively release recombinant proteins located in the cytoplasm of bacteria
and is recommended when lysing mammalian cells. However, this method for lysis is
not recommended when working with nitrilases as it has been noticed that purified
nitrilases suffer structural damages upon freezing.
Most animal tissues can be processed fresh (unfrozen). However, it is important to
keep them cold and to process them quickly (within 30 minutes) after dissection.
When disrupting fresh tissue, the cells need to be sheared immediately at the time the
GITC lysis solution is added. This can be done by dispensing the lysing solution in the
tube, adding the tissue and immediately sonicating. Samples should never be left
sitting in lysis solution, undisrupted. Hard tissues should be first treated in a blender
or a mechanical homogenizer.
Animal tissues that have been frozen after collection should be disrupted by grinding
in liquid nitrogen with a mortar and pestle. During this process, it is important that the
equipment and tissue remain at cryogenic temperatures. The tissue should be dry and
powdery after grinding. Grinding should be followed by sonication in a GITC lysis
buffer. Processing frozen tissue in this way is cumbersome and time consuming, but
effective.
Ultrasonic processing will typically cause the temperature of the sample to increase
especially with small volumes. Since high temperatures inhibit cavitation, the sample
temperature should be kept as low as possible - preferably just above its freezing
point. This can be accomplished by pulsing the ultrasonics on and off while keeping
the sample vessel immersed in an ice bath. While processing the sample occasionally
touch the vessel to ensure that the sample is relatively cool.
Increasing hydrostatic pressure (typically 15-60 psi) and viscosity can enhance cell
disruption. For microorganisms, the addition of glass beads in the 0.5 to 1mm size
range promotes cell disruption. Beads are almost a prerequisite when working with
spores and yeast. A good ratio is one volume of beads to two volumes of liquid. Glass
beads are available from:
Cole-Parmer Instruments Fisher Bioblock Scientific
625 East Bunker Court Parc d’innovation – BP 50111
Vernon Hills, Illinois 60061 F-67403 illkirch cedex
USA France
Phone: 1-800-323-4340 Phone: 03 88 67 14 14
Fax: 847-247-2929 Fax: 03 88 67 11 68
Email: [email protected] Email: [email protected]