Features

A fast way to purify plasmids for transfection

• Novel technology to diminish endotoxin content (< 50 EU/µg DNA) for successful transfection
• Purification in mini-format simplifies your work-flow
• Ultra-fast procedure

Applications

• Typical downstream applications: transfection of common cell lines (lipofection, electroporation)
• Also suitable for standard molecular biology application such as cloning and sequencing
  

Principle/Procedure

The NucleoSpin® 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 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 where plasmid DNA binds to the surface. Endotoxins and proteins are removed by the innovative Detoxification Buffer ERB. Further contaminations 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).

NucleoSpin® Plasmid Transfection-grade procedure

Application data

Plasmids were purified from E. coli (DH5α) with different plasmid purification products from MACHEREY-NAGEL.

Endotoxin levels appropriate for individual applications
A quantitative chromogenic LAL-test was used to assess endotoxin content. As indicated, the content of endotoxin is strongly depended on the technology of plasmid purification. Low endotoxin levels were detected after purification with NucleoSpin® Plasmid Transfection-grade, resulting in a plasmid solution directly appropriate for transfection of common cells.

Cell compatibility of eluted DNA

A pCMV-GFP plasmid (kindly provided by PlasmidFactory GmbH und Co. KG, Bielefeld, Germany) was purified from E. coli using NucleoSpin® Plasmid Transfection-grade. Plasmids were transfected into HEK239 cells by lipofection (Lipofectamine 2000) or nucleofection (Lonza) with > 90 % transfection ratio in both cases. Cell survival was compared to controls without DNA addition. Similar ratios show that cell-survival is not affected by DNA eluates purified with NucleoSpin® Plasmid Transfection-grade.

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