By P.C. van der Vliet (Eds.)
The primary position of RNA in lots of mobile techniques, in biotechnology, and as pharmaceutical brokers, has created an curiosity in experimental tools utilized to RNA molecules. This e-book offers scientists with a entire selection of completely established updated manuals for investigating RNA-protein complexes in vitro. The protocols could be played by way of researchers educated in general molecular organic thoughts and require no less than really good apparatus. The tactics comprise advice of providers of reagents.
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Additional info for Analysis of RNA-Protein Complexes in vitro
3'-end labelling of RNA 3'-end labelling of RNA can either be done by ligating [cx-~~P] pCp to the 3'-end using RNA ligase,I8 or adding a 3'-deoxynucleotide such as [a-32P]cordycepin-5'-triphosphateusing poly(A) polymerase. The advantage of the ligase method is that radioactive pCp is relatively easy to make from standard radioactive nucleotides. 1. RNA ligase method. 4) T 4 RNA ligase ( 5 U/pl) ATP, 10 mM Procedure 1. Dilute 5-20 pmol RNA in water. Adjust H20 so the total volume of the ligation will be 20 p1.
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.
Denature RNA at 95°C for 30 s, then on ice. 3. Add 40 kl phosphatase mix ( 5 pl 10 X CZP buffer, 35 PI HzO, 10 units Calf Intestinal Phosphatase). 4. Incubate at 37°C for 15 min followed by incubation at 56°C for 15 min. 5 . Add an additional 10 units of Calf Intestine Phosphatase. 6 . Repeat step 4. 7 . Add 2 pi 200 mM EGTA. 8. Heat inactivate at 95°C for 4 mins-then on ice. 9. 0. 10. " 11. Extract 2 X with chloroform. 12. 5 vol EtOH. 13. Precipitate and wash with 70% EtOH. Notes a. The heat inactivation does not completely inactivate the phosphatase.
Analysis of RNA-Protein Complexes in vitro by P.C. van der Vliet (Eds.)