The processing of a plasmid-based gene from E. Coli. Primary recovery by filtration

Abstract We describe the primary recovery of plasmid DNA from alkaline lysis mixtures using a nutsche filter operated under pressure. Six different filter cloths constructed of polypropylene, polyester and stainless steel were tested, with pore sizes ranging from 5–160␣μm. Both pore size and the material of the filter membranes employed in filtration experiments exerted considerable impact on the purity and yield of the plasmid DNA. The greatest degree of solids extrusion, shearing of chromosomal DNA and subsequent contamination of the filtrate was observed with the 160␣μm polyester filter. The best compromise was obtained with a 5␣μm polypropylene cloth. For an alkaline lysis mixture containing 101␣g wet weight solids per litre, filtration through this cloth proceeded at an average rate of 22.5␣cm␣h−1. Virtually complete removal of solids (99.4%) and protein (96.8%) was achieved, with a 8.2-fold purification of plasmid DNA at the expense of a 33% loss in yield. The filtration performance of this membrane was further modified by precoating with diatomaceous earths of different permeabilities (0.07–1.2␣darcies). The finest filter aid resulted in very pure plasmid DNA (65%), complete suspended solids removal and < 1% of the original protein remaining in the filtrate. However, the plasmid yield was only 30%, the processing rate was markedly reduced (8.2␣cm␣h−1), and some losses of plasmid DNA, due to adsorption on to the diatomaceous earth, were also observed (5.7%).