Endotoxins are copurified during plasmid preparations from bacterial lysates. Since they interfere with eukaryotic cell survival, endotoxin reduction is essential prior to cell transfection. Macherey-Nagel has developed a new column format and a novel buffer chemistry to enable vacuum-processed isolation of transfection-grade plasmid DNA in a midiprep format.
The NucleoSnap Plasmid Midi kits are designed for the rapid purification of highly pure plasmid DNA from up to 50 ml of a standard E. coli overnight culture. Plasmid DNA isolated with this kit is suitable for all common downstream applications, such as enzymatic digestion, cloning, sequencing, PCR amplification, transformation, and transfection (research use only).
E. coli cells are grown in a standard culture medium under appropriate selective conditions and harvested by centrifugation. Cells are resuspended in Resuspension Buffer SN1 and then lysed with Lysis Buffer SN2 containing sodium dodecyl sulfate and sodium hydroxide. Alkaline conditions ensure a complete and almost immediate denaturation of DNA and proteins. Addition of Neutralization Buffer SN3 precipitates potassium dodecyl sulfate complexes with bacterial cell debris, proteins, and macromolecular contaminants and neutralizes the pH, resulting in a reannealing of the covalently closed circular plasmid DNA, which remains soluble.
Debris is removed by a filtration step with the specially designed NucleoSpin Plasmid Filter Columns. The clear flowthrough contains plasmid DNA, while genomic DNA, cell remnants, and most of the proteins are filtered out and can be discarded.
The flowthrough containing the plasmid DNA is mixed with Precipitation Buffer SN4 and loaded into a NucleoSnap Plasmid Midi Column connected to a vacuum device. A vacuum is applied until the solution has completely passed through the filtration matrix. Endotoxins are washed away by Endotoxin Removal Buffer SN5, while salts and additional impurities are subsequently removed by a washing step with ethanolic Wash Buffer SN6.
Residual ethanol from Wash Buffer SN6 is efficiently removed by centrifugation in a microcentrifuge. To enable the use of a microcentrifuge, the NucleoSnap Columns are equipped with a predetermined breaking point and can be divided into a funnel component and a mini spin column by simply snapping off the funnel component. Plasmid DNA is eluted in Elution Buffer SNE (5 mM Tris/HCl, pH 8.5) and is ready for any common downstream application. No further clean-up steps are required.