By P.C. van der Vliet (Eds.)
The primary position of RNA in lots of mobile methods, in biotechnology, and as pharmaceutical brokers, has created an curiosity in experimental equipment utilized to RNA molecules. This booklet offers scientists with a complete selection of completely established up to date manuals for investigating RNA-protein complexes in vitro. The protocols will be played through researchers expert in regular molecular organic concepts and require at the least really expert apparatus. The strategies contain suggestion of providers of reagents.
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Extra resources for Analysis of RNA-Protein Complexes 'in vitro'
7 M Ch. 2 PREPARATION OF RNA 21 Equipment 50 ml polypropylene tubes (Falcon, Nunc) Polytron Ultra-ClearTM tubes (14 x 89 mm, Beckman) Ultracentrifuge and SW41 rotor Procedure 1. 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.
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. 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.
1~13mM Cp. H20 to 20 ~ 1 . 1 T 4 Polynucleotide kinase (5 U/pl). Incubate at 37°C for 30 min and heat-inactivate at 70°C for 5 min. Comment The [ ' ~ - ~ ~ P ]need p c p not be purified before use in the ligase reaction. 4. Specific modification of R N A at an internal site In many structural and functional studies it is desirable to modify a specific site of an RNA. Site-specific modifications at internal positions of small RNAs (<20 nucleotides) is most conveniently obtained by introducing a modified nucleotide during chemical synthesis by standard phosphoramidite chemistry and will not be covered in this book.