QIAfilter Cartridges (see figure “QIAfilter Mega-Giga Cartridge
”) are special filter units designed to replace the centrifugation step 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. Prepacked HiSpeed Tips operate by gravity flow and never run dry, minimizing the hands-on time required for plasmid preparation.
The unique QIAconcentrator (see figure “HighSpeed Plasmid Giga EF procedure
”) replaces the centrifugation step traditionally used to collect isopropanol-precipitated DNA following purification. The QIAconcentrator module traps the precipitated DNA, while the isopropanol-buffer mixture flows through. The DNA is then simply eluted from the QIAprecipitator into a collection tube with TE buffer or water. This unique module also eliminates the risk of pellet loss, which can occur during decanting of the supernatant following centrifugation.
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 ||Transfection, cloning sequencing, gene silencing ||Transfection, cloning sequencing, gene silencing |
|Culture volume/starting material ||500 ml culture volume ||2.5 l culture volume |
|Plasmid type ||High-copy, cosmid DNA ||High-copy, cosmid DNA |
|Processing ||Vacuum, centrifugation ||Vacuum, Centrifugation |
|Sample per run ||6 sample per run ||6 sample per run |
|Technology ||Anion-exchange technology ||Anion-exchange technology |
|Time per run ||50 min ||50 min |
|Yield ||2.5 mg ||10 mg |