Methods of enhancing the recovery of plasmid genes from neutralised cell lysate

In this study we have investigated the use of flotation and filtration, sin gly and combined, to enhance the separation of plasmid containing liquors f rom neutralised lysates with very different levels of solids. Filtration of crude neutralised lysates, containing roughly 100 g l(-1) solids, through various diatomaceous earth and cellulose precoat materials was invariably a ccompanied by severe loss of plasmid through adsorption and/or absorption. The use of more refined and inert filter aids did not alleviate these probl ems. The finest filter aid, Celatom FP-1SL, gave the best compromise of fil trate clarity (solids content of 0.05 g l(-1)) and plasmid purity (71%) and was selected for further studies involving combined use of flotation and f iltration. Removing the vast bulk of solids prior to filtration by flotatio n of the flee and draining of the plasmid liquor beneath, impacted dramatic ally on the filtration performance. Though systematic reductions in the sol ids challenge per unit filter area were accompanied by increased flux, elev ated levels of solids extrusion, chromosomal DNA and protein contamination were also observed, and losses of plasmid to filter aids were still high. W e have observed that increasing the scale of operation during lysis and neu tralisation from 0.3 or 0.6 l to 15 l is accompanied by significant improve ments in separation of cell debris solids from the plasmid and increased re coveries of the plasmid containing liquor. At the latter scale, the drained liquor contained similar to 80% of the plasmid and the solids content was only 0.2 g l(-1).