Endotoxins (lipopolysaccharides, or 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 and gene therapy applications is an endotoxin-free plasmid DNA.
Minimizing 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. Unlike competitor kits, there is no need for a time-consuming incubation step to remove the endotoxins (see procedure). The working procedure for the endotoxin-free plasmid preparation is identical to the standard protocol, and each prep yields up to 2,000 µg of endotoxin-free plasmid DNA.
NucleoBond EF kits employ 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. No additional incubation step is required for endotoxin removal. After efficient washing of the column, the purified plasmid DNA is eluted from the column.