Preparative purification of plasmid DNA templates for in vitro transcription assays by consecutive differential precipitations.
A procedure for large-scale isolation of plasmid DNA without the use of RNase has been developed to obtain a DNA template for preparative in vitro RNA synthesis catalyzed by phage RNA polymerases. The separation of plasmid DNA from admixtures has been achieved only through selective precipitations of either plasmid DNA or contaminants. No expensive reagents or equipment were required. Plasmid quality was evaluated by gel electrophoresis and restriction analysis. The obtained plasmid DNA templates have been shown to be devoid of any detectable ribonucleolytic activity that may interfere with the following RNA synthesis.
Stepanov
Victor G
VG
Institute of Molecular and Structural Biology, University of Aarhus, Denmark.
Nyborg
Jens
J
D011232:Chemical Precipitation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D010957:Plasmids / Q000096:biosynthesis* / Q000302:isolation & purification*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012313:RNA / Q000378:metabolism*; D013604:T-Phages / Q000235:genetics; D013698:Templates, Genetic; D014158:Transcription, Genetic
D012313:RNA