By P.C. van der Vliet (Eds.)
The valuable function of RNA in lots of mobile tactics, in biotechnology, and as pharmaceutical brokers, has created an curiosity in experimental equipment utilized to RNA molecules. This publication presents scientists with a complete selection of completely established updated manuals for investigating RNA-protein complexes in vitro. The protocols might be played through researchers informed in general molecular organic suggestions and require at least really expert apparatus. The techniques comprise suggestion of providers of reagents.
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Extra info for Analysis of RNA-Protein Complexes 'in vitro'
3. g. yeast). 0. 3 SDS 10% Phenol (saturated with acetate buffer) Equipment 50 ml polypropylene tubes Waterbath at 65°C 26 ANALYSIS OF RNA-PROTEIN COMPLEXES IN VITRO Heating block at 65°C Dry ice-ethanol bath Procedure 1. " 2. Discard the supernatant and wash the pellet in 1ml acetate buffer. Transfer to a microfuge tube and centrifuge for 3min at 4°C. 3. Discard the supernatant and resuspend the pellet in 500 p1 icecold acetate buffer. 4. Add 50 p1 10% SDS and mix briefly. 5. ' 6. Chill quickly to room temperature in a water bath and centrifuge for 5 min at room temperature.
5 . Add an additional 10 units of Calf Intestine Phosphatase. 6 . Repeat step 4. 7 . Add 2 pi 200 mM EGTA. 8. Heat inactivate at 95°C for 4 mins-then on ice. 9. 0. 10. " 11. Extract 2 X with chloroform. 12. 5 vol EtOH. 13. Precipitate and wash with 70% EtOH. Notes a. The heat inactivation does not completely inactivate the phosphatase. Comments The dephosphorylation of in vitro transcribed RNA can be avoided if a diribonucleotides, such as CpG, ApG or UpG is included in the transcription mixture.
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. M. (1996). RNaseH cleavage for processing of in vitro transcribed RNA for NMR studies and RNA ligation. RNA 2, 289-293.