By P.C. van der Vliet (Eds.)
The primary position 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 e-book offers scientists with a complete selection of completely proven updated manuals for investigating RNA-protein complexes in vitro. The protocols could be played via researchers educated in ordinary molecular organic concepts and require at least really good gear. The tactics comprise advice of providers of reagents.
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Extra resources for Analysis of RNA-Protein Complexes 'in vitro'
FEBS Lett. 215,327-330. 14. A. and Shetty, K. (1997). Defining the chemical groups essential for Tetrahymena group I intron function by nucleotide analog interference mapping. Proc. Natl. Acad. Sci. USA 94, 2903-2908. 15. K. and Krupp, G. (1995). 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.
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. Comments Since the guanidinium thiocyanate acid-phenol method can process Ch. 2 PREPARATION OF R N A 25 a large number of samples on a small scale, several manufacturers base their total RNA isolation kits on this method.
For crosslinking purposes, 5-bromo-U- or 5-iodo-U phosphoramidites are available. However, depending on the type of modification, the 5'-3'-coupling efficiency is typically decreased to 90-97% which Ch. 2 49 PREPARATION OF RNA DNA-ligase A. 5' M T d nucleotide 5'-RNA 3'-RNA 3' Bridging DNA-oligo B. DNA-ligase Bridging DNA-oligo Fig. 3. The principle behind site-specific modification of RNA. A, Ligation of two RNAs. In the illustrated example, the 3'-RNA contains a S'-phosphorylated modified nucleotide at the 5'-end.
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