Principle

The new generation of anion exchanger – ultra-fast, endotoxin-free plasmid DNA with high yield

Based on the innovative NucleoBond® Xtra product line which was introduced in early 2006 MACHEREY-NAGEL has now developed the endo-free version for ultra-fast plasmid preps with high yields of endotoxin-free DNA: NucleoBond® Xtra EF. NucleoBond® Xtra EF features up to 68% reduced prep times and up to 100% increased yields of endotoxin-free plasmid DNA compared to other commercially available kits for endotoxin-free plasmid purification. NucleoBond® Xtra EF is available in Midi and Maxi format as well as the ultra-fast Plus versions including NucleoBond® Finalizers. With NucleoBond® Xtra EF typical yields of ≥ 250 μg (Midi) or ≥ 1000 μg (Maxi) of ultra-pure plasmid DNA can be obtained. The kits contain enlarged NucleoBond® Xtra columns which leads to lower silica resin beds. This in turn enables faster flow of lysate and buffers through the columns. Specially designed column filters are included for convenient and time-saving clarification of bacterial lysates. The column filters are supplied inserted in the NucleoBond® Xtra columns and allow parallel clarification of bacterial lysate and loading onto the column. Their large, structured surface leads to high filter flow rates and minimized risk of clogging. The improved silica material with greater DNA binding capacity is based on the proven NucleoBond® anion-exchanger group MAE, methylaminoethanol (patented technology). Optimized buffer compositions lead to improved yields and highly efficient endotoxin removal.

Bacteria are harvested from an overnight culture and lysed by an optimized alkaline lysis procedure. The bacterial lysate is cleared and loaded onto the equilibrated column in one step (plasmid DNA binds to the anion-exchange resin). The first washing step using a special wash buffer is performed with inserted column filter to wash out residual lysate from the filter and obtain maximum recovery of DNA. After two subsequent washing steps using a buffer to eliminate endotoxins (Buffer ENDO-EF) and a wash buffer to remove contaminations (Buffer WASH-EF), the purified plasmid DNA is eluted in a high-salt buffer and precipitated with isopropanol. Separation of the precipitate can be performed by centrifugation (NucleoBond® Xtra Midi EF or Maxi EF) or by using the NucleoBond® Finalizer/Finalizer Large included in the NucleoBond® Xtra Midi Plus EF/Maxi Plus EF kits to reduce the total prep time to about 45 minutes (Midi Plus EF) or 50 minutes (Maxi Plus EF).

Features