( Kathleen A. Maguire-Zeis...)Commercial Plasmid Preparation Methods Yield DNA With Different Transfection Capabilities

Kathleen A. Maguire-Zeiss, Brandon K. Harv e y, Rita E. Giuliano and H o w a rd J. Federo ff, University of Rochester, Rochester, NY, USA

Introduction
Transfection efficiency is dependent upon a number of factors, including method of transfection, cell type, and DNA concentration and purity. Liposome-mediated transfection of mammalian cells has proven to yield higher transfection efficiencies and is more reproducible than other methods (Felgner et al. 1987; Rose et al. 1991). However, we have noted a significant difference in transfection efficiency among cell lines and among diff e rent DNA p reparations with liposomemediated transfections. We have sought to determine which method ensures optimal transfection eff iciencies . We prepared plasmid DNA by four commonly employed methods and measured its transfection efficiency in three different cell lines. DNA prepared using modified alkaline lysis followed by purification on Bio-Rads Quantum Prep DNA binding matrix gave superior transfection efficiencies in all three cell lines.

Method
DNA PREPARATION
DNA from the plasmid pHSVlac (Geschwind et al. 1994) was prepared using four different methods. For each method, pHSVlac DNA was prepared from pellets of overnight cultures (250500 ml, grown with antibiotic) by a modified alkaline lysis procedure with further purification according to each suppliers recommended protocol and buffer composition. Method A was a noncommercial reference method; we used the protocol of Saporito-Irwin et al. (1997), which purifies plasmid DNA by repeated precipitation with ammonium acetate . Method D was the protocol supplied by Bio-Rad with it
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