Description
The Ultrapure 100 Buffer Set is a set of buffers for use with the Ultrapure 500 Resin. Up to 100 mg endotoxin-free (<0.1 EU/µg DNA) plasmid DNA is easily and efficiently purified. The system uses QIAGEN anion-exchange technology with integrated endotoxin removal to yield ultrapure DNA.
With the Ultrapure 100 Buffer Set, up to 100 mg endotoxin-free plasmid DNA can be purified. The system uses QIAGEN anion-exchange technology with integrated endotoxin removal to yield ultrapure DNA. The exceptional separation properties result in DNA purity equivalent or superior to that obtained by two successive rounds of CsCl gradient centrifugation. The Ultrapure 100 Buffer Set provides buffers that are certified to contain <10 EU/ml (as determined by the BioWhittaker Kinetic-QCLTest) for use with the resin.
The level of endotoxin contamination in purified plasmid DNA depends on the purification method used. 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. The QIAGEN Ultrapure 100 system include an integrated endotoxin-removal step to yield plasmid DNA containing <0.1 EU/µg plasmid DNA. 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. 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.
The bacterial biomass is lysed under alkaline conditions and the lysate is cleared by centrifugation (see flowchart "Ultrapure 100 procedure"). Endotoxins are removed using the special Endotoxin Removal Buffer, and the lysate is loaded onto the Ultrapure 500 Resin for DNA purification.
