For purification of up to 500 µg endotoxin-free advanced transfection-grade plasmid or cosmid DNA

  • Less than 0.1 EU/µg DNA achieved
  • Fast procedure, with integrated endotoxin removal
  • High yields of high-copy plasmid DNA
  • LyseBlue for optimum lysis and maximum DNA yield

EndoFree Plasmid Kits provide rapid, anion-exchange-based endotoxin-free plasmid DNA purification. QIAfilter Cartridges enable fast lysate clearing by filtration. DNA yields up to 500 μg (Maxi), 2.5 mg (Mega), or 10 mg (Giga) are achieved. The purified DNA exceeds the purity obtained by 2 x CsCl gradient centrifugation and is suitable for advanced transfection-grade applications.

Quantity Product Cat. no. Price
 
 
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Quantity Product Cat. no. Price Sum
 
EndoFree Plasmid Maxi Kit (10)
10 QIAGEN-tip 500, Reagents, 10 QIAfilter Maxi Cartridges, Endotoxin-free Buffers
12362
$321.00
$0.00
The EndoFree Plasmid Maxi 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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Plasmid purification method versus transfection efficiency.|QIAGEN Plasmid Kit procedures.|Plasmid purity versus transfection efficiency.|Bacteria cell wall.|
Two independent pRSVcat preparations obtained with each method shown were each transfected twice into COS-7, HeLa, and LMH cells using a liposome-mediated method and into Huh7 cells using calcium phosphate. Average transfection efficiencies are expressed as percentages relative to the efficiency obtained with DNA prepared using the QIAGEN Plasmid Kit (100%).
|Neutralized bacterial lysates are incubated directly in the QIAfilter Cartridge and cleared in seconds by filtration. After the endotoxin removal step, 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.|Effect of the amount and quality of plasmid DNA on transfection efficiency in CHO SSF3 X9 cells grown in suspension under serum-free conditions is shown. Each point represents the mean of three independent experiments; Rel. LU: relative light units. (Data kindly provided by M. Zang-Gandor, Novartis AG, Basel, Switzerland.)
|Schematic diagram of the envelope of E. coli.|
Performance

The EndoFree Plasmid Maxi Kit integrates an efficient endotoxin removal step into the plasmid purification procedure — no additional extractions or affinity columns to remove lipopolysaccharides are required. Bacterial lysates are cleared by filtration with the QIAfilter Maxi Cartridge, and plasmid DNA is purified on gravity-flow QIAGEN-tip 500 anion-exchange tips. Up to 500 µg DNA can be purified from 100–250 ml culture. (Culture volumes depend on plasmid copy number, size of insert, host strain, and culture medium.) Purified DNA is endotoxin-free (<0.1 EU/µg DNA).

EndoFree Plasmid Kits remove bacterial endotoxins which are released during the lysis step and influence transfection of DNA into primary cells and sensitive cultured cells. The endotoxin-free DNA obtained from the EndoFree Plasmid Kits is highly suited for reproducible and reliable results in plasmid-mediated gene silencing, transfection (see figures "Plasmid purification method versus transfection efficiency" and "Plasmid purity versus transfection efficiency" and tables "Endotoxin levels in plasmid preparations" and "EndoFree DNA yields high transfection efficiencies with primary cells"). QIAGEN ultrapure endotoxin-free DNA is also suitable for gene therapy research and genetic vaccination and other sensitive applications.

Endotoxin levels in plasmid preparations*
Plasmid preparation method Endotoxin
(EU/µg DNA)
Average transfection
efficiency
EndoFree Plasmid Kit 0.1 154%
QIAGEN Plasmid Kit 9.3 100%
2x CsCl 2.6 99%
Silica-gel slurry 1230.0 24%
* Host strain: DH5α plasmid: pRSVcat.
1 ng LPS = 1.8 EU. 
Calculated from data in Figure "Plasmid purification method versus transfection efficiency".
EndoFree DNA yields high transfection efficiencies with primary cells*
DNA purification methodPercentage of transfected cells
EndoFree Plasmid Kit 21.0% ± 0.93
QIAGEN Plasmid Kit 8.1% ± 0.57
Silica-gel slurry 5.2% ± 0.74
* Primary rabbit gastric parietal cells were transfected with pEGFP-N2 (CLONTECH) prepared by the methods indicated. Transfections were performed using Effectene Transfection Reagent. The data represent the percentage of cells expressing GFP as determined by scoring the number of green fluorescent cells 48 hours post transfection. The transfection efficiencies represent the average from 6 to 9 replicate dishes from more than 2 different DNA preps for each purification method. (Data kindly provided by C. Chew and J. Parente, Department of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia, USA.)
Principle

The level of endotoxin contamination in purified plasmid DNA depends on the purification method used (see table "Endotoxin levels in plasmid preparations"). Silica-slurry–purified DNA exhibits extremely high endotoxin levels. QIAGEN, QIAfilter, and HiSpeed Plasmid Kits and 2x CsCl ultracentrifugation yield very pure DNA with relatively low levels of endotoxin. EndoFree Plasmid Kits include an integrated endotoxin-removal step to yield plasmid DNA containing <0.1 EU/µg plasmid DNA.

QIAfilter Cartridges, provided in QIAfilter, HiSpeed, and EndoFree Plasmid Kits, are special filter units designed to replace centrifugation following alkaline lysis of bacterial cells. QIAfilter Cartridges completely remove SDS precipitates and clear bacterial lysates in a fraction of the time needed for centrifugation, reducing plasmid-purification time by up to 1 hour. QIAfilter Maxi Cartridges have a syringe-format and lysates are cleared in a matter of seconds by pushing the liquid through the filter.

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.

 Endotoxins, also known as lipopolysaccharides or LPS, are cell-membrane components of Gram-negative bacteria such as E. coli (see figure "Bacterial cell wall"). Endotoxins are released during the lysis step of plasmid purification and significantly reduce transfection efficiencies in endotoxin sensitive cell lines (see figures "Plasmid purification method versus transfection efficiency" and "Plasmid purity versus transfection efficiency" and tables "Endotoxin levels in plasmid preparations" and "EndoFree DNA yields high transfection efficiencies with primary cells"). Furthermore, endotoxins can influence the uptake of plasmid DNA in transfection experiments by competing with DNA for “free” transfection reagent. Endotoxins also induce nonspecific activation of immune responses in immune cells such as macrophages and B cells, which can lead to misinterpretation of transfection results. These responses include induced synthesis of proteins and lipids such as IL-1 and prostaglandin. Overall, endotoxins represent a noncontrollable variable in transfection experiment setup, influencing the outcome and reproducibility of results and making them difficult to compare and interpret. In gene therapy research, endotoxins can interfere by causing endotoxic-shock syndrome and activation of the complement cascade. 

Procedure

Bacterial cells are lysed under alkaline conditions and the crude lysates are cleared using the QIAfilter Maxi Cartridge. At this stage, the Endotoxin Removal Buffer is added to the filtered lysate, which is incubated on ice. 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 (see flowchart "QIAGEN Plasmid Kit procedures"). The DNA is concentrated and desalted by isopropanol precipitation and collected by centrifugation.

Applications

DNA purified with EndoFree Plasmid Kits is suitable for any sensitive application, including:

Transfection, including primary, sensitive, and suspension cells 
Gene therapy research
Gene silencing
Microinjection
Feature
Specifications
Applications Transfection, gene therapy
Culture volume/starting material 100 ml – 250 ml culture volume
Plasmid type High-copy, low-copy, cosmid DNA
Processing Manual (vacuum)
Samples per run; throughput 1 sample per run
Technology Anion-exchange technology
Time per run or prep per run 150 min
Yield <500 µg

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Supplementary Protocols
2
This protocol is for purification of up to 100 µg endotoxin-free plasmid DNA using QIAGEN-tip 100.
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Endotoxin-free DNA is essential for gene therapy research and will improve transfection into sensitive eukaryotic cells.
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