The E.Z.N.A.^® Fastfilter Endo-free Plasmid Maxiprep Kit integrates an efficient endotoxin removal step into the E.Z.N.A.^® Fastfilter^® plasmid procedure. It offers a simple, rapid, and cost effective method for purifying high quality, endotoxin-free plasmid (0.1 EU/µl ) for high efficiency transfection.

Principle

The E.Z.N.A.^® Fastfilter Endo-Free Plasmid Maxiprep Kit combines the power of HiBind™ technology with the time-tested consistency of alkaline-SDS lysis of bacterial cells to deliver high quality DNA with lower level of endotoxin. HiBind™ matrix that avidly, but reversibly, binds DNA under certain optimal conditions allowing proteins and other contaminants to be removed. Nucleic acids are easily eluted with de-ionized water or low salt buffer. This kit also include a special filter cartridge, which replaces the centrifugation step following alkaline lysis. Following the lysis, the cleared lysate are extracted with ETR Buffer which will almost completely remove the endotoxin. Then the lysate is applied to the HiBind™ Maxi-columns , plasmid DNA is bound to the silica membrane and contaminants are removed with a simple wash step. The use of maxi-columns facilitate the binding, washing, and elution steps thus enabling multiple samples to be simultaneously processed. For more information on columns, click Flexible Multi-Format Columns.

Procedure

Bacterial cells are harvested from overnight culture by centrifugation and lysed by a modified alkaline-SDS lysis procedure. After addition of Neutralization Buffer, the precipitates are removed with the FastFilter^® unit to produce a cleared lysate. Endotoxins are removed from the cleared lysate with simple extraction phase-separation steps. The sample is then applied to a HiBind^® DNA Maxi column. After a few quick wash steps, purified DNA can be eluted with low salt buffer or water.

Applications

E.Z.N.A.^® Fastfilter Endo-free Plasmid Maxiprep Kit provides high quality, reproducible plasmid DNA for use in the following applications :

• Automated fluorescent DNA sequencing
• Transfection of Mammalian cells
• Restriction Digestion