For purification of up to 10 mg transfection-grade plasmid or cosmid DNA
ABI PRISM 7000 (TaqMan) analysis of 1 µg samples of a 105 kb BAC DNA construct isolated using either the QIAGEN Large-Construct Kit or a standard anion-exchange procedure is shown. The lower level of contaminating genomic DNA with the QIAGEN Large-Construct Kit is indicated by the greater number of amplification cycles required for its detection.
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.