By P.C. van der Vliet (Eds.)
The primary function of RNA in lots of mobile methods, in biotechnology, and as pharmaceutical brokers, has created an curiosity in experimental tools utilized to RNA molecules. This e-book presents scientists with a finished number of completely validated up to date manuals for investigating RNA-protein complexes in vitro. The protocols should be played by way of researchers informed in general molecular organic thoughts and require at the very least really good apparatus. The methods contain suggestion of providers of reagents.
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Additional info for Analysis of RNA-Protein Complexes in vitro
A, Ligation of two RNAs. In the illustrated example, the 3'-RNA contains a S'-phosphorylated modified nucleotide at the 5'-end. B, Ligation of three RNAs. The short centrally positioned RNA, which can be synthesised chemically, contains the modified nucleotide. In both examples, the RNAs are aligned by using a bridging DNA oligonucleotide, and the ligation(s) is catalysed by T4 DNA ligase. means that only a fraction of RNAs larger than 15-20mers is chemically homogeneous. This method is therefore not useful for large RNAs.
9. Transfer the upper aqueous phase to a new tube, taking care to avoid the interphase. 10. Add 400 p1 chloroform and extract vigorously. Centrifuge for 5 min. 11. Transfer the aqueous layer to a new microfuge tube. d Notes a. 1 and Note b below). b. The yield of extracted RNA can be improved by including 2 g acid-washed glass beads. c. A combined heating block and shaker such as the Eppendorf 5436 thermomixer is useful. Ch. 2 PREPARATION OF RNA 27 d. 4) followed by an additional ethanol precipitation.
Add 2 p1 lox Polynucleotide kinase buffer and mix. 5 . Add 1 pl T4 Polynucleotide kinase ( 5 U/pl). 6 . Incubate at 37°C for 30 min, then on ice. 7. 5 M EDTA. 8. b 9. Precipitate and wash with 70% EtOH. Notes a. Lyophilise [ Y - ~ ~ATP P ] if dissolved in ethanol solution. b. Precipitation is optional if gel filtration methods are used for the purification. Comments The end-labelled RNA can be purified in a denaturing polyacrylamide gel (n < 1000) or an agarose gel ( n > 1000) or by gel filtration in Sephadex G-50.
Analysis of RNA-Protein Complexes in vitro by P.C. van der Vliet (Eds.)