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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.
NucleoSpin® Plasmid procedure
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Detection of virulent plasmids from Bacillus anthracis using artificial contaminated sample material (e.g., soil samples or various meals and powders) with a positive control strain
(pXO1+, pXO2–). After deaden of spores via H2O2 in a liquid culture the resulting pellet is used for plasmid DNA preparation with a slightly modified protocol of the NucleoSpin® Plasmid kit.
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A: Plasmid DNA was isolated according to the procedure mentioned above and used in LightCycler. analysis using the LightCycler™ Bacillus anthracis Detection Kit (Roche Diagnostics). The different CT values are caused by varying spike levels (5 x 102 - 103 CFU/mL). No inhibition of PCR reactions is observed.
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B: Shows corresponding melting curves from the slopes in Figure A. The equal melting point of 60.55°C for all fragments verifies that only one fragment size has been amplified.
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C: From the above performed LightCycler™ PCR 15 μL were loaded on a 1.5% TAE gel. For each spiked sample one distinct lane is visible which backs up the results of the melting curve in Figure B. The PCR product has a size of 329 bp.
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M: 100 bp marker
-: negative control
+: positive control
lanes 1-8: spiked samples
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All data shown was kindly provided by Dr. Beyer, Institut für Umwelt- und Tierhygiene, Universität Hohenheim, Germany
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High-purity plasmid mini prep
Agarose gel analysis of pUC19 plasmid DNA after purification with the NucleoSpin® Plasmid kit. 2 mL of E. coli DH5a™. culture grown in LB medium (lane 2), 5 mL culture (lane 3) and 8 mL culture (lane 4) were processed; lane 1: marker
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High-quality DNA for automated sequencing
Dye-terminator sequence of vector pBluescript® SK– purified with the NucleoSpin® Plasmid kit. Sequence was analyzed on a MegaBACE 1000 DNA Analysis System.
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Click for downloading
the sequencing profile
as pdf file
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