NucleoBond 96 Xtra EF, the high-throughput kit of the NucleoBond Xtra product line, is designed for isolating high-quality, endotoxin-free plasmid DNA in a 96-well plate format. Depending on the culture conditions and growth medium, approximately 2–4 µg of plasmid DNA (from a 96-well plate culture) or up to 50 µg of plasmid DNA (from a glass tube incubation) can be isolated per well. Growing cultures 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-exchange group MAE (methylaminoethanol; patented technology).
In the NucleoBond 96 Xtra EF procedure, E. coli cells are lysed using SDS/alkaline lysis. Cell lysates are clarified using a NucleoBond Filter Plate and then loaded onto the equilibrated NucleoBond Xtra EF Plate for plasmid binding. After subsequent washes with buffers to eliminate endotoxins and other contaminants, plasmid DNA is eluted in a high-salt buffer and precipitated with isopropanol. Precipitate separation is achieved using a specially-designed NucleoBond Finalizer Plate, eliminating the need for a time-consuming centrifugation step.
The kit can be used with vacuum or centrifugation processing. The NucleoBond Xtra EF Plate is used under gravity flow to achieve a specific flow rate needed for efficient binding to the anion exchange group.
Required hardware for vacuum processing and centrifugation
Processing NucleoBond 96 Xtra EF under vacuum requires using the NucleoVac 96 Vacuum Manifold. Centrifuge processing requires using 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 can reach accelerations of 5,600–6,000 x g (bucket height: 85 mm). For further information about suitable centrifuges, please contact Technical Support.