Co-isolation of in vivo32P-labeled specific transcripts and DNA without phenol extraction or nuclease digestion

Abstract

A method is described for isolation and quantitation of specific intact transcripts, for which a hybridization probe is available, from 32P-labeled bacterial cells. The RNA is extracted in the absence of RNase activity by incorporating an inert, physically removable RNase inhibitor throughout the spheroplasting, cell lysis, and pronase digestion steps. [ 32P]RNA is separated from [ 32P]DNA, without recourse to phenol extraction or DNase treatment, on a Cs 2SO 4HCONH 2 step gradient in which the precipitated RNA forms a sharp band. Specific transcripts are purified from [ 32P]RNA by physical separation of the transcript and hybridization probe using gel-exclusion chromatography. The gentleness of this technique enables the co-isolation of DNA and can facilitate the analysis of covalently joined RNA-DNA replication intermediates.