Endotoxins (lipopolysaccharides, LPS) are a major component of the Gram-negative bacterial cell wall. The LPS molecule is an extremely potent stimulator of the mammalian immune system, and a number of mechanisms exist to detect LPS and to respond to the presence of either this molecule or Gram-negative bacteria. LPS is a common contaminant of plasmid DNA preparations grown in E. coli. The negative charges associated with lipid A and the inner core of LPS cause the LPS molecule to behave like DNA on anion exchange chromatography resins. An important prerequisite for transfections is an endotoxin-free plasmid DNA.
The level of endotoxin contamination in purified plasmid DNA is critical for many applications. For this reason MACHEREY-NAGEL developed a specific patented procedure that reduces endotoxins to a very low level. In comparison to competitor kits, there is no need for a time-consuming incubation step to remove the endotoxins. The working protocol for endotoxin-free plasmid preparation, which is identical to the standard protocol, employs a modified alkaline/SDS lysis procedure to prepare the bacterial cell pellet for plasmid purification. The bacterial lysate is cleared by filtration and loaded onto the equilibrated column. After efficient washing of the column, the purified plasmid DNA is eluted from the column.
NucleoBond Endotoxin-Free Plasmid Gigaprep Kits allow you to purify up to 9,000 µg of endotoxin-free plasmid DNA (<0.1 EU/µg DNA) in just a few hours without phenol/chloroform extraction. The kits can be used to purify high-copy-number plasmids from E. coli. The resulting plasmids can be used for applications where the level of endotoxin contamination is critical, such as transfection of highly sensitive cells, as well as PCR, restriction analysis, and lentivirus production.