Analysis of RNA-Protein Complexes in vitro by P.C. van der Vliet (Eds.)
By P.C. van der Vliet (Eds.)
The relevant position of RNA in lots of mobile strategies, in biotechnology, and as pharmaceutical brokers, has created an curiosity in experimental tools utilized to RNA molecules. This publication offers scientists with a finished number of completely confirmed updated manuals for investigating RNA-protein complexes in vitro. The protocols will be played by means of researchers informed in usual molecular organic innovations and require at least really expert gear. The approaches comprise advice of providers of reagents.
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Extra resources for Analysis of RNA-Protein Complexes in vitro
Means that only a fraction of RNAs larger than 15-20mers is chemically homogeneous. This method is therefore not useful for large RNAs. Site-specific modification of large RNAs (>20 nucleotides) has until recently been a difficult task which involved an RNA ligasemediated ligation of two RNAs. An alternative method has been developed by Moore and Sharp'' which has facilitated this reaction. The basic principle is outlined in Fig. 3A: Two RNAs, a 5'RNA and a 3'-RNA, containing the sequences 5' and 3' of the site to be modified, respectively, are synthesised so that the 3'-RNA contains the 5'-phosphorylated modified nucleotide at the 5'-end.
The integrity of the isolated RNA can be judged from denaturing agarose gel electrophoresis followed by ethidium bromide staining, since the 28 S ribosomal RNA band ought to exhibit a considerably greater fluoresecens than that obtained from the 18 S ribosomal RNA band. Nevertheless, placenta and pancreas Ch. 2 PREPARATION OF RNA 23 with their high ribonuclease contents are difficult to work with, and liver contains large amounts of glycogen that may lead to a slight contamination of the RNA preparation.
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. Nucleic Acids Res. 18, 837-844. 17. S. and Krupp, G. (1992). Tetrahedron Lett. 33, 3301-3304.