The NucleoSpin Plasmid Miniprep Kit is designed for the rapid (25 min), high-yield (up to 40 µg), small-scale preparation of high-purity plasmid DNA. These miniprep columns contain a unique silica membrane that binds up to 40 µg of plasmid DNA. The purified DNA is suitable for transfection, cloning, sequencing, PCR, transformation, and restriction analysis.
The miniprep kit allows purification of up to 25 μg plasmid DNA per preparation, from 1–5 ml of saturated E. coli culture. You can also use this kit to purify up to 40 μg of plasmid DNA from 5–10 ml E. coli culture. In order to obtain optimal results from 5–10 ml E. coli culture, increase your buffer volumes using the NucleoSpin Buffer Set.
Using the NucleoSpin Plasmid Miniprep Kit
Harvested bacteria are resuspended and processed by SDS/alkaline lysis (see the User Manual, located in the Documents tab below). High-salt buffer is added to neutralize the lysate and to create appropriate conditions for DNA binding to the silica membrane. After centrifugation, the clear supernatant is loaded onto a NucleoSpin Plasmid miniprep spin column.
Contaminants like salts and macromolecular cellular components are removed by simple washing with ethanolic Buffer A4. Additional washing with Buffer AW is recommended for the following demands:
- Complete removal of high levels of endonucleases, with buffer AW prewarmed to 50°C (e.g. from wild-type E. coli strains)
- Processing 5–10 ml of bacterial culture
- Long read length in DNA sequencing
- Better performance of critical enzymatic reactions
Highly pure plasmid miniprep DNA is finally eluted in 50 μl slightly alkaline Buffer AE (5 mM Tris/HCl, pH 8.5). Alternatively, water (pH 8.0–8.5) can be used.
