Clontech/A fast way to purify plasmids for transfection in 96-well plates—NucleoSpin 96 Plasmid Transfection-grade/96 Preps/740491.1

The NucleoSpin 96 Plasmid Transfection-grade kit uses a new technology developed by Macherey-Nagel to reduce the level of endotoxins copurified 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. Processing can be performed manually or on automated liquid handling platforms. A miniprep format is also available.

Principle/procedure

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).

Automation

The NucleoSpin 96 Plasmid Transfection-grade protocol can be fully automated on common liquid handling platforms using vacuum processing. Please contact Technical Support to confirm compatibility of the kit with your automation platform.

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 (either plate or tube centrifuge) is required.

Figure 1. The NucleoSpin 96 Plasmid Transfection-grade procedure.

Application data

Figure 2. NucleoSpin 96 Plasmid Transfection-grade provides higher yields of transfection-grade plasmid DNA than a competitor. Plasmid DNA was isolated from 3 ml of an E. coli culture (A₆₀₀=2) with the NucleoSpin 96 Plasmid Transfection-grade kit and a 96-well format kit from Competitor Z. DNA yield was measured by absorption readings.

Figure 3. NucleoSpin 96 Plasmid Transfection-grade yields higher-quality plasmid DNA than a competitor. Plasmid DNA was isolated from 3 ml of an E. coli culture (A₆₀₀=2) with the NucleoSpin 96 Plasmid Transfection-grade kit and a 96-well format kit from Competitor Z. DNA purity was assessed by absorption readings. An A_260/A280 ratio of 1.8 was obtained for plasmid DNA isolated with the NucleoSpin 96 Plasmid Transfection-grade kit, indicating the absence of contaminating compounds (e.g., RNA or proteins).

Figure 4. The NucleoSpin 96 Plasmid Transfection-grade kit provides reproducible yields. High-copy plasmid DNA was isolated from 3 ml of an E. coli culture with the NucleoSpin 96 Plasmid Transfection-grade kit, and the eluates were loaded on a 1% TAE agarose gel and stained with ethidium bromide. Lane M: 1 kb ladder (Thermo Fisher Scientific). Lanes 1–23: Eluted plasmid samples.