The NucleoSpin 96 Plasmid Transfection-grade kit uses a new technology developed by Macherey-Nagel to reduce the level of endotoxins co-purified during plasmid preparations from bacterial lysates, in a 96-well-plate format. Since endotoxins interfere with eukaryotic cell survival, endotoxin reduction is essential prior to cell transfection.
The NucleoSpin 96 Plasmid Transfection-grade procedure is a modified version of the Birnboim and Doly alkaline lysis plasmid miniprep protocol. Pelleted bacteria are resuspended in Buffer A1 and plasmid DNA is liberated from the cells by SDS/alkaline lysis with Buffer A2. Buffer A3 neutralizes the lysate; precipitates genomic DNA, proteins, and cell debris; and creates appropriate conditions for binding of plasmid DNA to the silica membrane.
The crude lysate is cleared by centrifugation and brought into contact with a silica membrane which binds plasmid DNA on its surface. Endotoxins and proteins are removed by the innovative Detoxification Buffer ERB. Further contaminants such as salts are removed with ethanolic Buffer AQ, while traces of ethanol are removed by centrifugation.
Pure plasmid DNA is eluted under low ionic strength conditions with slightly alkaline Buffer AE (5 mM Tris/HCl, pH 8.5) and is ready for any common downstream application, including transfection (research use only).
Required hardware for vacuum processing and centrifugation
The NucleoSpin 96 Plasmid Transfection-grade kits can be used manually with the NucleoVac 96 Vacuum Manifold. Additionally, a suitable centrifuge for harvesting the bacteria (plate or tube centrifuge) is required.