By P.C. van der Vliet (Eds.)
The significant 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 accomplished choice of completely verified updated manuals for investigating RNA-protein complexes in vitro. The protocols might be played by means of researchers educated in typical molecular organic ideas and require not less than really expert apparatus. The tactics contain advice of providers of reagents.
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Extra info for Analysis of RNA-Protein Complexes 'in vitro'
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.
5 ~1 H2O. 3. 1). 4. Mix and incubate at 37°C for 1 h. 5. 0). 6. Add 1 pl 5 mg/ml tRNA ( E . coli RNase free) or glycogen ( 5 mg/ml) if tRNA is critical. 7. Extract 1 x with phenol. 8. Extract 1 x with chloroform. 9. Add 500 pl EtOH and precipitate. 10. Wash with 70% EtOH. 11. Redissolve in H20. Notes a. 1. b. RNA labelled to a high specific activity is unstable and should be used within a day if full length RNA is required. c. Similar procedures can be used €or other modified nucleotides. 2). d.
Repeat wash 2-3 times. 6. Wash twice in H 2 0 for 30 s each time. 7. Rinse in EtOH, dry and count (count B). Assuming equal representation of the different nucleotides. 24 x (count B/count A) x (nmols cold UTP in the reaction). 2. Cotranscriptional labelling or modiJication of RNA Radioactive or modified nucleotides can be incorporated randomly in a transcript or specifically at the 5’-position of the transcript during transcription. Thus it is feasible to incorporate cap-structures 36 ANALYSIS OF RNA-PROTEIN COMPLEXES IN VITRO (GpppG), dinucleotides (NpG), terminal monophosphate (pG, thio-G) and photoreactive nucleotides at the 5’-end.
Analysis of RNA-Protein Complexes 'in vitro' by P.C. van der Vliet (Eds.)