Purification of plasmid DNA by tangential flow filtration.
A simple, scalable method for purification of plasmid DNA is described. The method includes modification of the classical alkaline-lysis-based plasmid extraction method by extending the solubilization step from less than 30 min to 24 h. The extraction is followed by the novel use of tangential flow filtration (TFF) for purification of the remaining contaminants. The method does not include the use of any organic solvents, RNase, high-speed centrifugation, or column chromatography steps. The method typically yields 15 to 20 mg of plasmid DNA per liter of bacterial culture and results in removal of >99% of RNA and >95% of the protein that remains after the modified alkaline lysis procedure. The procedure has been demonstrated to be effective in the isolation of seven different plasmids. Plasmids isolated using this method had comparable transfection capability relative to plasmid isolated using a classical, cesium chloride gradient-based method.
Kahn
D W
DW
Department of Recovery Science, Genentech, Inc., 1 DNA Way, South San Francisco, California, USA. kahn.david@gene.com
Butler
M D
MD
Cohen
D L
DL
Gordon
M
M
Kahn
J W
JW
Winkler
M E
ME
D002460:Cell Line; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D005169:Factor VIII / Q000235:genetics / Q000378:metabolism; D005374:Filtration / Q000379:methods*; D006801:Humans; D010957:Plasmids; D014162:Transfection
D005169:Factor VIII; D004247:DNA