By adding a quick endotoxin removal step to the E-Z 96^® Mag-Bind^® Plasmid Isolation protocol, the E-Z 96^® Mag-Bind^® Endo-free Plasmid Isolation Kit provides a flexible and fast solution for isolating endotoxin free plasmid DNA on a small, economical scale. This flexible system is easily automated making it an ideal option for high throughput transfection screening.

Principle

The E-Z 96^®Magbead^® Endo-free Plasmid Isolation Kit combines the power of MagBead^® technology with the time-tested consistency of alkaline- SDS lysis of bacterial cells to deliver high quality endotoxin free plasmid DNA. Magbead^® Particle avidly, but reversibly, binds DNA under certain optimal conditions allowing proteins and other contaminants to be removed. Nucleic acids are easily eluted with deionized water or low salt buffer. By using 96-well format, up to 96 samples can be simultaneously processed in less than 60 minutes. The new E-Z 96^® Lysate Clearance Plate obviates time-consuming centrifugation for clearing of the bacterial alkaline lysates. Following the lysis, the cleared lysate are extracted with ETR Buffer which will almost completely remove the endotoxin.

Procedure

Bacterial cells grown in 96 deep well plate are pelleted by centrifugation and re-suspended in Solution I/RNase A. Add Solution II to obtain a cleared lysate and neutralization buffer and mix well until a flocculent white precipitate forms. Cell lysates are then cleared with E-Z 96^® Lysate Clearance Plate. Endotoxins are removed from the cleared lysate with simple extraction phase-separation steps. The endotoxin free cleared lysate is then transferred to the 96-well Binding plate and Mag-Bind^® Particles Solution E and MGC Binding Buffer is added. After mixing and incubation, Plasmid DNA are selectively bound to MagBead Particles. Wash the Mag-Bind^® particles twice with SPM wash buffer, air-dry them and elute the purified DNA with water or TE Buffer.

Applications

E-Z 96^® Magbead^® Endo-free Plasmid Isolation Kit provides high quality, reproducible plasmid DNA for use in the following applications :

• High throughput transfection screening