For purification of up to 100 µg transfection-grade plasmid or cosmid DNA

  • Purity equivalent to 2 x CsCl gradient centrifugation
  • High yields of plasmid DNA
  • Cost-effective preparations
  • LyseBlue for optimum lysis and maximum DNA yield

QIAGEN Plasmid Kits provide gravity-flow, anion-exchange tips for purification of transfection-grade plasmid DNA. The QIAGEN Plasmid Midi Kit delivers DNA yields of up to 100 μg. Lysate clearing and isopropanol precipitation are achieved by centrifugation.

Quantity Product Cat. no. Price
 
 
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Quantity Product Cat. no. Price Sum
 
QIAGEN Plasmid Midi Kit (25)
25 QIAGEN-tip 100, Reagents, Buffers
12143
$265.00
$0.00
 
QIAGEN Plasmid Midi Kit (100)
100 QIAGEN-tip 100, Reagents, Buffers
12145
$982.00
$0.00
The QIAGEN Plasmid Midi Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Transfection efficiency versus plasmid purification method.|QIAGEN Plasmid Kit procedures.|
Different pRSVcat DNA preparations using the methods indicated were introduced into the indicated cell lines by liposome-mediated transfection, and the efficiencies determined by measuring CAT expression levels after 40 h. Each bar represents the mean of 4 independent transfections (2 transfections with each of 2 independent plasmid preparations). The highest transfection efficiency was achieved with QIAGEN Plasmid Kits.
|Neutralized bacterial lysates are cleared by centrifugation. The cleared lysate is then loaded onto the anion-exchange tip where plasmid DNA selectively binds under appropriate low-salt and pH conditions. RNA, proteins, metabolites, and other low-molecular-weight impurities are removed by a medium-salt wash, and ultrapure plasmid DNA is eluted in high-salt buffer. The DNA is concentrated and desalted by isopropanol precipitation and collected by centrifugation.
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Performance
Classic QIAGEN Plasmid Midi Kits use gravity-flow QIAGEN-tip 100 anion-exchange tips for efficient purification of plasmid DNA. Up to 100 µg high-copy plasmid DNA is purified from 25–100 ml culture. (Culture volumes depend on plasmid copy number, size of insert, host strain, and culture medium.) Plasmid DNA purified with QIAGEN Plasmid Kits is highly suitable for use in applications such as transfection (see figure "Transfection efficiency versus plasmid purification method"), cloning, sequencing, and in vitro transcription.
Principle

The unique anion-exchange resin in QIAGEN-tips is developed exclusively for the purification of nucleic acids. Its exceptional separation properties result in DNA purity equivalent or superior to that obtained by two successive rounds of CsCl gradient centrifugation. Prepacked QIAGEN-tips operate by gravity flow and never run dry, minimizing the hands-on time required for plasmid preparation. The entire QIAGEN plasmid purification system avoids the use of toxic substances such as phenol, chloroform, ethidium bromide, and CsCl, minimizing hazard both to the user and the environment.

Procedure

With QIAGEN Plasmid Kits, bacterial lysates are cleared by centrifugation. The cleared lysate is then loaded onto the anion-exchange tip where plasmid DNA selectively binds under appropriate low-salt and pH conditions. RNA, proteins, metabolites, and other low-molecular-weight impurities are removed by a medium-salt wash, and pure plasmid DNA is eluted in high-salt buffer (see flowchart "QIAGEN Plasmid Kit procedures"). The DNA is concentrated and desalted by isopropanol precipitation and collected by centrifugation. 

Applications

Plasmid DNA purified with QIAGEN Plasmid Kits is highly suited for use in applications, such as:

Transfection
Cloning
PCR
In vitro transcription
Feature
Specifications
Applications Transfection, cloning, sequencing, capillary sequencing etc.
Culture volume/starting material 25–100 ml culture volume
Plasmid type High-copy, low-copy, cosmid DNA
Processing Manual (centrifugation)
Samples per run; throughput 1 sample per run
Technology Anion-exchange technology
Time per run or prep per run 150 min
Yield <100 µg

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User-Developed Protocols
10
The procedure has been used successfully for isolation of high- and low-copy-number plasmids from various Bacillus subtilis strains. Yield of plasmid DNA was typically 10-20 µg plasmid DNA from 100 ml culture.
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The procedure has been used successfully for isolation of high-copy-number plasmids from Citrobacter freundii. Yield of plasmid DNA was typically 3-8 µg DNA per ml culture.
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The procedure has been used successfully for isolation of high-copy-number plasmids from Proteus vulgaris and Proteus mirabilis. Yield of plasmid DNA was typically 3-8 µg DNA per ml culture.
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The procedure has been used successfully for isolation of cryptic plasmids (pLC2-based) from mesophilic Lactobacillus strains such as L. sake and L. curvatus. Yield of plasmid DNA was typically 10-20 µg plasmid DNA from 100 ml culture.
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This procedure has been used successfully for isolation of 150-250 kb BAC DNA from a mouse-BAC library cloned in pBeloBAC11 from Escherichia coli strain HB101/r. The yield of BAC DNA from 100 ml culture was typically 20-40 μg.

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The procedure has been used successfully for isolation of 110 kb P1 DNA (pAdsacBII with an 80 kb insert) from Escherichia coli strain NS3529. Yield of P1 DNA was typically 10-50 µg from 500 ml culture.
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The procedure has been used successfully for isolation of a variety of medium-copy-number shuttle vectors from S. xylosus, S. carnosus, S. epidermidis, and S. aureus. Yield of plasmid DNA was typically 2-10 µg from 50 ml culture.
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The procedure has been used successfully for isolation of the large (128 kb), very-low-copynumber (1-2 copies per cell) plasmid pHCG3 and its derivatives from Oligotropha carboxidovorans. Yield of plasmid DNA was typically 3-6 µg plasmid DNA from 200 ml culture.
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The procedure has been used successfully for isolation of linear plasmids from Borrelia burgdorferi sensu lato species, which include Borrelia burgdorferi sensu stricto, Borrelia afzelli, and Borrelia garinii.
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