By P.C. van der Vliet (Eds.)
The vital position of RNA in lots of mobile tactics, in biotechnology, and as pharmaceutical brokers, has created an curiosity in experimental tools utilized to RNA molecules. This publication presents scientists with a accomplished choice of completely demonstrated up to date manuals for investigating RNA-protein complexes in vitro. The protocols may be played through researchers knowledgeable in usual molecular organic suggestions and require at the least really good apparatus. The approaches contain suggestion of providers of reagents.
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Extra resources for Analysis of RNA-Protein Complexes 'in vitro'
9. 5 ml rnicrofuge tube. 10. Precipitate with 300 pl isopropanol at -20°C for 1 h. 11. Centrifuge at 10,000 g for 20 min at 4°C and discard the supernatant. 12. d 13. Dissolve the dried pellet in 20 pl HzO. Notes a. 1% antifoam A. b. Do not use neutralised phenol. c. The volume is unlikely to exceed 1ml. d. The ethanol precipitation step is introduced to remove traces of guanidinium thiocyanate. Comments Since the guanidinium thiocyanate acid-phenol method can process Ch. 2 PREPARATION OF R N A 25 a large number of samples on a small scale, several manufacturers base their total RNA isolation kits on this method.
Leave on ice for 15 min and centrifuge at 10,000 g for 10 rnin at 4°C. 6 . Avoid the interphase and transfer the aqueous phase' to a 2 ml microfuge tube. 7. Add an equal volume of isopropanol, mix and place at -20°C for 1 h. 8. Centrifuge at 10,000 g for 20 min at 4°C and discard the supernatant. 9. 5 ml rnicrofuge tube. 10. Precipitate with 300 pl isopropanol at -20°C for 1 h. 11. Centrifuge at 10,000 g for 20 min at 4°C and discard the supernatant. 12. d 13. Dissolve the dried pellet in 20 pl HzO.
Redissolve the dried pellet in 100 ~1 double-distilled and autoclaved H20. 20. Estimate the amount and the purity by measuring both the AZboand the A2so. Notes a. Use eye-protection during the crushing and do not allow the pieces to thaw. Transfer the tissue pieces in liquid nitrogen into the polypropylene tube, and allow the liquid nitrogen to evaporate. Then add the lysis solution immediately. b. If insoluble material from tissue samples is present at this stage it should be removed by filtering through a Whatman 54 filter.
Analysis of RNA-Protein Complexes 'in vitro' by P.C. van der Vliet (Eds.)