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


Achieve superior transfection efficiencies with StrataPrep midiprep kit

Scott Basehore Jeff Braman
Stratagene

Plasmid DNA was purified with Stratagenes StrataPrep EF plasmid midiprep kit, a kit based on anion exchange chromatography, magnetic beads, and cesium chloride density gradient centrifugation. Plasmid yields and purity (as judged by 260-nm to 280-nm optical density ratios) were comparable with the four methods. However, endotoxin levels of the StrataPrep kit- and ion-exchange-purified plasmid DNA were nearly 800 times lower than magnetic bead-purified DNA and three times lower than DNA purified by cesium chloride density gradient centrifugation. The StrataPrep-purified plasmid yielded the highest efficiency transfection of an endotoxin-sensitive cell line when compared to the other three methods.

The StrataPrep EF plasmid midiprep kit is a simple and convenient method for obtaining up to 350 g of high-purity plasmid DNA from a single midi-spin cup device.1 Contaminating endotoxin may be easily removed by using the kits unique endotoxin removal buffer (ERB). Endotoxin, a common contaminant of plasmid DNA isolated from gram-negative E. coli hosts, is difficult to remove2; its presence in plasmid preparations lowers the transfection efficiency of endotoxin-sensitive cell lines2,3 and complicates the interpretation of experiments designed to examine signal transduction pathways triggered by a measured addition of endotoxin.4 Stratagenes unique ERB contains a color indicator to monitor endotoxin removal, thereby facilitating the purification process. Following endotoxin removal, plasmid DNA is bound to a special glass fiber matrix in a StrataPrep Kit midi-spin cup. Contaminants are removed by adding wash solution to the spin cup, centrifugi
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