Analysis of RNA-Protein Complexes 'in vitro' by P.C. van der Vliet (Eds.)

By P.C. van der Vliet (Eds.)

The principal position 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 accomplished choice of completely verified up to date manuals for investigating RNA-protein complexes in vitro. The protocols should be played by means of researchers expert in average molecular organic thoughts and require at the least really expert gear. The systems comprise advice of providers of reagents.

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The medium-salt wash improves the purity of poly-A+ RNA and is a potential source of mRNAs with oligo-A tails. d. The majority of poly-A+ RNA is eluted with 1ml elution buffer. Comments The amount of purified poly-A+ RNA is often so small that quantification by measurements in a standard spectrophotometer is too wasteful. Instead the purified poly-A+ RNA can be coelectrophoresed with total RNA and subjected to Northern analysis with a probe for an abundant mRNA such as eEF-la or GAPDH. Moreover, the Northern analysis has the added advantage of examining the integrity of the purified poly-A+ RNA.

Wash with 15 ml wash buffer. 3. 5. 4. Equilibrate the column with 10 ml 1 x Ioad buffer. 5 . Dilute the RNA sample to 250 p1 with H 2 0 and leave at 65°C for 5 min. 6. Add 250 $ 2 x load buffer and apply the sample to the column. 7. Collect the flow-through, leave it at 65°C for 5 min and reapply to the column. Ch. 2 8. 9. 10. 11. 12. 13. ' Elute poly-A+ RNA with 2 ml elution bufferd and place on ice. Extract with phenol-chloroform and ethanol-precipitate. Redissolve in 50 pl double-distilled HzO and store at -80°C.

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. 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.

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