Neutralized bacterial lysates are cleared by centrifugation. The cleared lysate is then loaded onto the anion-exchange tip where plasmid DNA selectively binds under appropriate low-salt and pH conditions. RNA, proteins, metabolites, and other low-molecular-weight impurities are removed by a medium-salt wash, and ultrapure plasmid DNA is eluted in high-salt buffer. The DNA is concentrated and desalted by isopropanol precipitation and collected by centrifugation. ..
Different pRSVcat DNA preparations using the methods indicated were introduced into the indicated cell lines by liposome-mediated transfection, and the efficiencies determined by measuring CAT expression levels after 40 h. Each bar represents the mean of 4 independent transfections (2 transfections with each of 2 independent plasmid preparations). The highest transfection efficiency was achieved with QIAGEN Plasmid Kits.