Sequencing-grade plasmid DNA
For rapid small scale preparation high-purity plasmid DNA
• Fast procedure – 6 Minipreps in 25 min
• High capacity – up to 50 μg of plasmid DNA
• Chaotropic salt wash buffer – extra pure DNA
• Silica-membrane technology
• Sample material: 2–10 mL E. coli culture
• Vector Size: < 50 kbp
• Typical yield: 25–45 µg
• Ratio A260/A280: 1.80–1.85
- Kit contents :
- NucleoSpin:registered: Plasmid Columns, Collection Tubes (2 mL), buffers, RNase A
- Reference numbers :
- 10 preps: 740588.10
- 50 preps: 740588.50
- 250 preps: 740588.250
- High-copy plasmids from E. coli
- Low-copy plasmids, P1 constructs, or cosmids (increased buffer volumes required, see “Ordering information”
- Plasmid DNA from Gram-positive bacteria and plasmid DNA clean-up from reaction mixtures
- Supplementary protocol for M13 DNA available on request, please contact Technical Service
- Typical downstream applications: cloning, sequencing, PCR, transformation, restriction analysis
NucleoSpin® Plasmid is designed for the rapid, small scale preparation of high-purity plasmid DNA. The kit allows purification of up to 25 μg plasmid DNA per preparation from 1–5 mL of a saturated E. coli culture. Up to 40 μg of plasmid DNA can be obtained when processing 5–10 mL E. coli culture.
For this purpose, the buffer volumes are increased using the NucleoSpin® Buffer Set in addition. Optimal results are obtained for isolation of plasmid DNA from E. coli strains. Harvested bacteria are resuspended and processed by SDS/alkaline lysis (see procedure). 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 spin column.
Contaminations 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 E. coli wild-type strains)
• Processing of 5–10 mL of bacterial culture
• Long read length in DNA sequencing
• Better performance of critical enzymatic reactions.
• Highly pure plasmid 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.
- PLoS One. 2008;3(11):e3647. doi: 10.1371/journal.pone.0003647. Epub 2008 Nov 5.
A one pot, one step, precision cloning method with high throughput capability.
Engler C, Kandzia R, Marillonnet S.