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.
| EndoFree Plasmid Maxi Kit Specifications | |||
| Expected yield* | Column capacity | Culture volume† | QIAfilter format |
| Up to 500 µg | 500 µg | 100–250 ml | Syringe-format Maxi Cartridge |
* Actual yields depend on plasmid copy number, size of insert, host strain, culture medium, and culture volume.
† Culture volumes depend on plasmid copy number, size of insert, host strain, and culture medium.
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 vs. Transfection Efficiency" and "Plasmid Purity vs. 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 non-controllable 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.
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 Midi and Maxi Cartridges have a syringe-format (see figure "QIAfilter Cartridge") 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. Pre-packed 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.
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 "Comparison of QIAGEN Plasmid Kit Procedures"). The DNA is concentrated and desalted by isopropanol precipitation and collected by centrifugation.
EndoFree Plasmid Kits yield endotoxin-free DNA which is ideal for high reproducibility and efficiency in plasmid-mediated gene silencing, transfection (see figures "Plasmid Purification Method vs. Transfection Efficiency" and "Plasmid Purity vs. 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 (see "Ultrapure 100 Column") 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: DH5 α plasmid: pRSVcat | †1 ng LPS = 1.8 EU | ‡Calculated from data in Figure "Plasmid Purification Method vs. Transfection Efficiency" |
EndoFree DNA yields high transfection efficiencies with primary cells
| 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.) | ||||||||||