Two independent pRSVcat preparations obtained with each method shown were each transfected twice into COS-7, HeLa, and LMH cells using a liposome-mediated method and into Huh7 cells using calcium phosphate. Average transfection efficiencies are expressed as percentages relative to the efficiency obtained with DNA prepared using the QIAGEN Plasmid Kit (100%).
Neutralized bacterial lysates are incubated directly in the QIAfilter Cartridge and cleared in seconds by filtration. After the endotoxin removal step, 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.
Effect of the amount and quality of plasmid DNA on transfection efficiency in CHO SSF3 X9 cells grown in suspension under serum-free conditions is shown. Each point represents the mean of three independent experiments; Rel. LU: relative light units. (Data kindly provided by M. Zang-Gandor, Novartis AG, Basel, Switzerland.)
