Sonics VC100 100-watt (1992) User Manual download pdf (Page 16)

If enzymes cannot be used, the following procedures should be considered: Freezing the

sample at -70?C overnight, then thawing it in water immediately prior to ultrasonic

processing.

Most animal tissues can be processed fresh (unfrozen). It is important to keep fresh tissue

cold and to process it quickly (within 30 minutes) after dissection. When working with

fresh tissue, the cells must be sonicated 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. Large samples of 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 thorough sonication in a GITC

lysis buffer. Processing frozen tissue in this way is cumbersome and time consuming, but

effective.

Cultured cells are normally easy to disrupt. Cells grown in suspension are collected by

centrifugation, rinsed with PBS to remove culture medium, and then lysed by sonicating

in a GITC lysis buffer. Placement of the vessel on ice while washing and lysing the cells

will further protect the RNA from endogenous RNases released during the disruption

process.

Soft, fresh plant tissue can often be disrupted by sonicating in a lysis buffer. Other plant

tissues, like pine needles, need to be ground dry, without liquid nitrogen. Some hard,

woody plant materials require freezing and grinding in liquid nitrogen prior to being

ultrasonically processed. Plant cell suspension cultures and calluses can typically be

sonicated in a lysis buffer within 2 minutes. The diversity of plants and plant tissue make

it impossible to give a single recommendation for all. However, most plant tissues

typically contain polysaccharides and polyphenols that can coprecipitate with RNA and

inhibit downstream assays. Treating a plant tissue lysate with polyvinylpyrrolidone

(PVP) will precipitate such problematic components from the lysate before the actual

RNA isolation is carried out.

Whenever possible, the tissues should be diced very small to permit movement within the

liquid. Tough tissues such as skin and muscle should be macerated first in a blender or

the like for about 10 seconds, and confined to a small vessel during ultrasonic treatment.

If sub-cellular particles are desired intact, the amplitude should be kept low, and the

processing time increased.

Yeast can be extremely difficult to disrupt because their cell walls may form capsules or

nearly indestructible spores. To process yeast, sonicate in a tube containing the sample,

guanidinium-based lysis buffer and small glass beads (0.5 – 1 mm). Pretreatment with

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Sonics VC100 100-watt (1992) User Manual Page 16