Sequential CaCl2, polyethylene glycol precipitation for RNase-free plasmid DNA isolation

Abstract

Functional genomics is facilitated by the ability to express genes in heterologous systems. In some cases function can be assayed by generation of in vitro transcripts of the unknown genes and expressing those transcripts in various expression systems. Plasmids bearing phage promoters are used to generate in vitro transcripts. Therefore, it is important to ensure that the template plasmid DNA is not contaminated with RNase from the isolation procedure. We have developed a plasmid purification protocol that does not utilize RNase yet yields pure plasmid DNA. The protocol combines the selective precipitation of RNA with 1.4M CaCl2, followed by a final selective precipitation of the plasmid DNA in a 10% polyethylene glycol (PEG), 250 mM NaCl solution. Purity of the resulting plasmid DNA was determined spectrophotometrically and by gel electrophoresis. No detectable contaminating RNA was observed in the plasmid DNA preparations. Inhibitory effects of the protocol were assayed by performing restriction analyses, sequencing, PCR, and in vitro transcription. These procedures were successful. The in vitro transcripts visualized by gel electrophoresis were found to be full length, thus indicating no significant endogenous RNase activity associated with the procedure.