By P.C. van der Vliet (Eds.)
The important function of RNA in lots of mobile methods, in biotechnology, and as pharmaceutical brokers, has created an curiosity in experimental equipment utilized to RNA molecules. This e-book presents scientists with a entire number of completely demonstrated updated manuals for investigating RNA-protein complexes in vitro. The protocols may be played through researchers expert in general molecular organic innovations and require at the very least really good apparatus. The methods comprise advice of providers of reagents.
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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. Some transcripts require nucleotides different from pppG at the 5'-end, and in these cases special precautions should be taken.
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. After use, regenerate the oligo(dT)-cellulose resin with 10 ml wash buffer, 10 ml HzO and 10ml 1 X load buffer.
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. 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.