High-throughput isolation of endotoxin-free plasmid DNA in 96-well format

Principle

The NucleoBond® 96 Xtra EF product is the high-throughput kit of the NucleoBond® Xtra product line. The kit provides 96 preparations of high-quality and endotoxin-free plasmid DNA. Depending on the cultivation conditions and growth medium, around 2–4 µg (96-well plate cultivation) or up to 50 µg (glass tube incubation) plasmid DNA can be isolated per well. Cultivation in rich media, such as Circlegrow® or TB, is recommended. The isolated plasmid DNA is of highest purity and can be used for transfection, even for sensitive cell lines such as neuronal cells.
The kit technology is based on anion-exchange chromatography using improved silica material with greater DNA binding capacity, based on the proven NucleoBond® anion-exchanger group MAE (methylaminoethanol; patented technology).
In the procedure, E. coli cells are lysed by a SDS/alkaline lysis. Cell lysates are clarified by the NucleoBond® Filter Plate and afterwards loaded onto the equilibrated NucleoBond® Xtra EF Plate for plasmid binding. After subsequent washing steps using buffers to eliminate the endotoxins and other contaminants, plasmid DNA  is eluted in a high-salt buffer and precipitated with isopropanol. Precipitate separation is achieved by the special NucleoBond® Finalizer Plate omitting the time-consuming centrifugation step.
The kit can be used by vacuum or centrifugation processing. Only the NucleoBond® Xtra EF Plate is used under gravity flow to realize a specific flow rate, needed for efficient binding to the anion exchanger group.
 
 
Required hardware

Vacuum processing
Processing NucleoBond® 96 Xtra EF under vacuum requires the NucleoVac 96 Vacuum Manifold.
 
Centrifugation processing
A microtiter plate centrifuge that is able to accommodate the NucleoBond® Filter / Finalizer Plates stacked on a Square-well Block or a Rack of Tube Strips and reaches accelerations of 5,600–6,000 x g (bucket height: 85 mm).

 Features