Qiagen, 11910, Ultrapure 100 Buffer Set

Buffers P1, P2, P3, QBT, QC, QN, ER, TE, endotoxin-free water, RNase A; for preparation of up to 100 mg plasmid DNA

Features

- Purification of <0.1 EU/µg plasmid DNA
- Purification of ultrapure plasmid DNA up to 100 mg

Product Details

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.

Performance

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.

Principle

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.

Applications

- Plasmid-mediated gene silencing
- Transfection
- Gene therapy research

The Ultrapure 100 Buffer Set with the Ultrapure 500 Resin yields plasmid DNA of a purity at least equivalent to that obtained after 2x CsCl gradient centrifugation. The simple endotoxin-removal step ensures that the purified DNA contains <0.1 EU/µg DNA. The DNA is therefore suitable for sensitive applications, including: