Application & Usage | E. coli cells are harvested, resuspended in Cell Resuspending Buffer (E1) with RNase A, and then lysed with Lysis Buffer (E2). Precipitation Buffer (E3) is added to the lysate and the lysate is clarified by centrifugation. The cleared lysate is passed through a pre-packed anion exchange column. The negatively charged phosphates on the backbone of the DNA interact with the positive charges on the surface of the resin. The temperature, salt concentration, and pH of the solutions influence binding. Under moderate salt conditions, plasmid DNA remains bound to the resin while RNA, proteins, carbohydrates and other impurities are washed away with Wash Buffer (E5). The plasmid DNA is eluted under high salt conditions with the Elution Buffer (E6). The eluted DNA is desalted and concentrated with an alcohol precipitation step. The entire protocol can be completed in 1.5–2 hours. |
