By P.C. van der Vliet (Eds.)
The critical 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 presents scientists with a finished selection of completely demonstrated up to date manuals for investigating RNA-protein complexes in vitro. The protocols might be played by means of researchers knowledgeable in commonplace molecular organic recommendations and require at least really good apparatus. The strategies contain suggestion of providers of reagents.
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Additional info for Analysis of RNA-Protein Complexes in vitro
However, the LiCl precipitation will jeopardise the yield of RNAs <200 nucleotides. 4. 5 10 mM MgClz 1 mM EDTA 10 units DNase I (RNase-free from Pharmacia). 3% SDS. Equipment Swinging bucket microfuge Procedure 1. Rinse the dish (100mm) twice with 10ml ice-cold PBS. 2. Add 1ml of PBS containing 2 mM EDTA, wait 30 s, and then scrape off cells into an Eppendorf tube. Spin for 10 s at 12,000 rpm and wash pellet with 1ml PBS. 3. Resuspend cells in 300 pl lysis buffer. Vortex gently for 10 s and leave on ice for 1min.
6. Wash twice in H 2 0 for 30 s each time. 7. Rinse in EtOH, dry and count (count B). Assuming equal representation of the different nucleotides. 24 x (count B/count A) x (nmols cold UTP in the reaction). 2. Cotranscriptional labelling or modiJication of RNA Radioactive or modified nucleotides can be incorporated randomly in a transcript or specifically at the 5’-position of the transcript during transcription. Thus it is feasible to incorporate cap-structures 36 ANALYSIS OF RNA-PROTEIN COMPLEXES IN VITRO (GpppG), dinucleotides (NpG), terminal monophosphate (pG, thio-G) and photoreactive nucleotides at the 5’-end.
1987). Sequence dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H. 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.