A method for production of 13C/15N double labelled RNA in E. coli, and subsequent in vitro synthesis of ribonucleotide 5' triphosphates.
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.
Nyholm
T
T
Unit for Molecular Genetics, Karolinska Institute, Huddinge, Sweden.
Andäng
M
M
Hotchkiss
G
G
Härd
T
T
Baumann
H
H
Larsson
S
S
Ahrlund-Richter
L
L
D002247:Carbon Isotopes; D002701:Chloramphenicol / Q000494:pharmacology; D017046:Cost Savings; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D009587:Nitrogen Isotopes; D010710:Phosphates / Q000737:chemistry* / Q000302:isolation & purification; D012329:RNA, Bacterial / Q000096:biosynthesis* / Q000187:drug effects; D012265:Ribonucleotides / Q000737:chemistry* / Q000302:isolation & purification; D013997:Time Factors
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D010710:Phosphates; D012329:RNA, Bacterial; D012265:Ribonucleotides; D002701:Chloramphenicol