Download Analysis of RNA-Protein Complexes 'in vitro' by P.C. van der Vliet (Eds.) PDF

By P.C. van der Vliet (Eds.)

The important function of RNA in lots of mobile approaches, in biotechnology, and as pharmaceutical brokers, has created an curiosity in experimental tools utilized to RNA molecules. This ebook presents scientists with a complete choice of completely verified up to date manuals for investigating RNA-protein complexes in vitro. The protocols could be played by means of researchers expert in average molecular organic ideas and require at the very least really expert apparatus. The tactics contain advice of providers of reagents.

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Adherent cells (about 10') can be lysed directly in the culture flasks whereas cells in suspension culture must be pelletted. " 2. 5 ml guanidinium thiocyanate lysis solution. 3. Homogenise at full-speed for 1 min with a Polytron. 4. 35 g sodium N-lauroylsarcosinate. Cap and invert the tube a few times-leave for 5-10 min. 5. b 6. Place 2 ml CsCl cushion at the bottom of a polyallomer ultracentrifuge tube (14 x 89 mm). 7. Layer the homogenate carefully on top of the cushion. 8. ' 9. Remove the majority of the supernatant with a 10 ml disposable pipette taking care not to touch the bottom of the tube.

The yield of extracted RNA can be improved by including 2 g acid-washed glass beads. c. A combined heating block and shaker such as the Eppendorf 5436 thermomixer is useful. Ch. 2 PREPARATION OF RNA 27 d. 4) followed by an additional ethanol precipitation. However, the LiCl precipitation will jeopardise the yield of RNAs <200 nucleotides. 4. 5 10 mM MgClz 1 mM EDTA 10 units DNase I (RNase-free from Pharmacia). 3% SDS. Equipment Swinging bucket microfuge Procedure 1. Rinse the dish (100mm) twice with 10ml ice-cold PBS.

8. F. C. (1989). Synthesis of small RNAs using 'I7 polymerase. Meth. Enzymol. 180, 51-62. 9. , Pokrovskaya. A. A. (1991). Preparatine in v i m mRNA synthesis using SP6 and l7 polymerases. Anal. Biochem. 195, 207-213. 10. A. A. (1984). Recognition of cap structure in splicing in vitro of mRNA precursors. Cell 38, 731-736. 11. Yu, Y. A. (1997). A new strategy for introducing photoactivatable 4-thiouridine ("U) into specific positions in a long RNA molecule. RNA 3, 807-810. 12. Lapham, J. and Crothers.

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