Sonics VCX750 User Manual download pdf (Page 26)

the sample remains trapped in intact cells and, therefore, is unavailable for

subsequent purification. For most samples, thorough disruption can be monitored by

close inspection of lysate after disruption. There should be no visible particulates,

except when disrupting materials containing hard, non-cellular components, such as

connective tissue or bone.

When processing difficult cells, pretreatment with an enzyme to “weaken” the cell

walls is beneficial. Lysozyme, byaluronidase, glycosidase, glucalase, lyticase,

zymolase and lysostaphin digestion are among the enzymatic methods frequently

used with yeast. Lysozyme, zymolase and lysostaphin digestion are among the

enzymatic methods frequently used with bacteria and yeast to dissolve a coat,

capsule, capsid or other structure not easily sheared by ultrasonics. Enzymatic

treatment is usually followed by sonication in a GITC lysis buffer. Collogenase may be

used with collogen, lysostaphin with staphylococcus, and trypsin hyaluronidase with

liver and kidney.

Yeast can be extremely difficult to disrupt. To process yeast sonicate in a tube

containing the sample, guanidinium-based lysis buffer and small glass beads (0.5 – 1

mm). Enzymatic pretreatment as outlined above is strongly recommended.

To disrupt filamentous fungi, scrape the mycelial mat into a cold mortar, add liquid

nitrogen, grind to a fine powder with a pestle, then sonicate in lysis buffer to solubilize

completely. As fungi may also be rich in polysaccharides, pretreatment with

polyvinylpyrrolidone (PVP) may be beneficial.

Disruption of cells found in soil and sediments is accomplished by 1) isolating the

bacterial cells from the material prior to the RNA isolation procedure. This is

accomplished by homogenization of wet soil in a mechanical blender followed by a

slow speed to pellet the bacteria cells. From this point, cells can be lysed as

described above for bacteria, or 2) isolating RNA from the soil or sediment directly.

For example, adding soil to a diatomaceous earth and lysis buffer, and then

sonicating. The sample is then centrifuged to remove solid debris.

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

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

GITC lysis buffer. Placing the flask or plate on ice while washing and lysing the cells

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

process.

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Sonics VCX750 User Manual Page 26