A fast way to purify plasmids for transfection in 96-well plates—NucleoSpin 96 Plasmid Transfection-grade
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.
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 A260/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.
Overview
- Novel technology to diminish endotoxin content (<50 EU/µg DNA) for successful transfection
- Innovative MN Wash Plate minimizes risk of cross-contamination
- Ultra-fast procedure
- Processing can be done under vacuum or by centrifugation
- Workflow can be fully automated on common liquid handling platforms
- NucleoSpin 96 Plasmid Transfection-grade Core Kits provide basic content focusing on automation platforms (additional accessories can be combined as needed).
More Information
| Technology | Silica membrane and endotoxin-reduction technologies |
| Format | 96-well plate |
| Processing | Manual or automated, vacuum or centrifugation |
| Endotoxin content | <50 EU/µg DNA (EU=Endotoxin Units) |
| Starting material | Up to 5 ml bacterial culture (E. coli, high-copy plasmids) |
| Vector size | <15 kb |
| Typical yield | 5–20 µg |
| A260/A280 | 1.80–1.85 |
| Elution volume | 100–200 µl |
| Preparation time | 45 min/plate |
| Binding capacity | 35 µg |
Applications
- Manual and automated isolation of high-copy plasmids
- Typical downstream applications: transfection of common cell lines, cell-based assays, screenings
Additional product information
Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.
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Tip for a successful transfection—remove endotoxins!
Endotoxins are copurified during plasmid preparations from bacterial lysates and affect eukaryotic cell survival, ultimately leading to a lower transfection efficiency. Innovative mini-format technology reduces endotoxins in your plasmid prep, leads to more successful transfections, and simplifies your workflow.
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