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 booklet offers scientists with a accomplished number of completely established updated manuals for investigating RNA-protein complexes in vitro. The protocols may be played via researchers proficient in average molecular organic suggestions and require no less than really expert apparatus. The techniques comprise suggestion of providers of reagents.
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Phosphate modijications: T7 polymerase does not discriminate significantly between NTP and [as]NTP (NEN). To obtain the appropriate modification level the NTP is substituted with [as]NTP to the desired level. 1, can be used. Concurrent base and phosphate modijication: The current examples of cotranscriptional incorporation of modified bases are rather limited,I4-l6 and the modified NTPs are generally not commercially available. g. 5% incorporation of inosine at the G positions requires 40% inosinetriphosphates in the reaction mixture, while 7-methyl-guanosine is incorporated as readily as GTP.
8. ' 9. Remove the majority of the supernatant with a 10 ml disposable pipette taking care not to touch the bottom of the tube. Pour off the remainder of the supernatant and leave the tube in an inverted position. 10. Cut off the bottom of the tube with a heated scalpel. 11. 1 autodigesteddproteinase K solution and transfer to a microfuge tube. 1 of proteinase K solution and combine in the microfuge tube. 12. Leave for 1 h at 37°C. 13. Extract with 2 x 300 ~1 phenol/chloroform and 300 pl chloro- 22 ANALYSIS OF RNA-PROTEIN COMPLEXES IN YITRO form transferring the aqueous phase to a new microfuge tube after each extraction.
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. The phenol and chloroform extraction should be included after the gel filtration.