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Highest Transfection Efficiency of an Endotoxin-Sensitive Mammalian Cell,,,Line

ng the cup, and eluting the purified DNA from the matrix in low-ionic-strength buffer. The resulting purified plasmid DNA is ready for subsequent molecular biology applications.

Four different plasmid purification methods were analyzed. Stratagene's StrataPrep EF plasmid midiprep kit, Qiagen's QIAfilter Plasmid Maxi kit with additional Endotoxin Free Buffer set (AEC: anion exchange chromotography), Promega's Wizard PureFection Plasmid DNA Purification system (MB: magnetic beads), and cesium chloride density gradient centrifugation (CsCl).

Yields and Purity

pCMV b-gal plasmid DNA was isolated from Epicurian Coli XL1-Blue MRF E. coli cells with the StrataPrep kit. Yields of plasmid DNA ranged from 360 g using the MB method to 473 g for CsCl. Yields of the StrataPrep Kit and AEC-purified DNA were 392 g and 433 g, respectively, for an equivalent volume of bacterial culture (Table 1).

The 260-nm to 280-nm optical density (OD) ratios ranged between 1.8 and 1.9 for each plasmid preparation (Table 1), indicating a high level of purity.5 OD values at 240 nm were also measured to detect other contaminants such as EDTA6 and detergents. The 260-nm to 240-nm OD ratios ranged between 1.5 and 1.7, which indicated that the plasmid preparations were essentially devoid of these contaminants6 (Table 1).

Endotoxin units per microgram of plasmid DNA (EU/g) were measured (BioWhittaker, Inc.) using the Kinetic QCL assay. Endotoxin levels for the StrataPrep Kit and AEC-purified DNA were approximately 800 times lower than MB-purified DNA and 3 times lower than CsCl-purified plasmid DNA (Table 1).

Transfection of Endo
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