PMID: 7541814Feb 1, 1995Paper

A method for production of 13C/15N double labelled RNA in E. coli, and subsequent in vitro synthesis of ribonucleotide 5' triphosphates

Journal of Biochemical and Biophysical Methods
T NyholmL Ahrlund-Richter

Abstract

In this paper we describe an enhanced method for the large scale production of high quality 13C/15N labelled NTPs. High amounts of labelled RNA was obtained from E. coli cells grown in 13C/15N enriched medium and treated with chloramphenicol. Total RNA was extracted from spheroplasted cells in the presence of SDS and proteinase K and subsequently degraded to NMPs by nuclease P1 and high concentrations of nuclease S1 in a low salt buffer. To avoid non-specific degradation of the RNA, nuclease digestion was performed in a short term reaction on native, not heat-denatured RNA. CMP, AMP, GMP and UMP were chromatographically separated and converted to the corresponding NTPs by a mixture of kinases in the presence of a coupled redox system based on thioredoxin and dithiothreitol. The quality of the 13C/15N labelled NTPs was tested by in vitro transcription.

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Citations

Nov 1, 2011·Journal of Biomolecular NMR·Chandar S Thakur, T Kwaku Dayie
Nov 18, 2011·Journal of Biomolecular NMR·Chandar S Thakur, T Kwaku Dayie
Dec 7, 2011·Journal of Biomolecular NMR·Barbara KrähenbühlGerhard Wider

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