Endotoxins are lipopolysaccharides included in the cell wall of gram-negative bacteria. Endotoxins are co-purified during standard plasmid preparations resulting in lower survival rate of transfected eukaryotic cells.
MACHEREY-NAGEL has developed a new technology to reduce endotoxins from bacterial lysates in a convenient silica membrane based preparation. The NucleoSnap Plasmid Midi kit is based on alkaline lysis to liberate plasmid DNA from the bacterial cells. The kit includes a NucleoSpin® Plasmid Filter Column for lysate clarification by centrifugation. Endotoxins and proteins are removed during the wash step by the innovative Detoxification Buffer. Additionally, the kit is based on a new innovative column design composed of an upper part that can hold up to 30 mL E.coli lysate and a bottom part which is a mini spin column for DNA concentration. After loading the cleared lysate and the wash steps with the large volumes on a vacuum manifold, the upper part can be snapped of and the mini spin column can be processed in a table top centrifuge. The mini spin column allows Plasmid DNA elution in 200 – 500 µL elution buffer without the need of time consuming DNA precipitation.
Advantages: MACHEREY-NAGEL introduces a new column design that allows the isolation of up to 700 µg plasmid DNA out of 50 mL E.coli culture within 35 minutes. Furthermore, the NucleoSnap Plasmid Midi kit introduces a novel technology to diminish endotoxin content for successful transfections of standard cell lines like HEK293 or CHO.