By P.C. van der Vliet (Eds.)
The principal function of RNA in lots of mobile techniques, in biotechnology, and as pharmaceutical brokers, has created an curiosity in experimental tools utilized to RNA molecules. This booklet offers scientists with a complete choice of completely demonstrated up to date manuals for investigating RNA-protein complexes in vitro. The protocols should be played by way of researchers proficient in commonplace molecular organic options and require at least really expert gear. The systems comprise suggestion of providers of reagents.
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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.
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. " Excess of the cap analogue does not affect the elongation of the transcript al- 38 ANALYSIS OF RNA-PROTEIN COMPLEXES IN VITRO though the reduction in GTP concentration will reduce the overall efficiency of the transcription. Comments The transcription efficiency is greatly reduced or abolished if the 5'nucleotide is not a guanosine.
Enzymatic synthesis of 2'-modified nucleic acids: identification of important phosphate and ribose moieties in RNase P substrates. Nucleic Acids Res. 23, 18451853. 16. ,Char, S. and Krupp, G. (1990). The methylation of one specific guanosine in a pre-tRNA prevents cleavage by RNase P and by the catalytic M1 RNA. Nucleic Acids Res. 18, 837-844. 17. S. and Krupp, G. (1992). Tetrahedron Lett. 33, 3301-3304.
Analysis of RNA-Protein Complexes 'in vitro' by P.C. van der Vliet (Eds.)