Large-scale purification of plasmid DNA by fast protein liquid chromatography using a Hi-Load Q Sepharose column.
The large-scale purification of plasmid DNA was achieved using fast protein liquid chromatography on a Hi-Load Q Sepharose column. This method allows for the purification of plasmids starting from crude plasmid DNA, prepared by a simple alkaline lysis procedure, to pure DNA in less than 5 h. In contrast to the previously described plasmid purification methods of CsCl gradient centrifugation or high-pressure liquid chromatography, this method does not require the use of any hazardous or expensive chemicals. More than 100 plasmids varying in size from 3 to 15 kb have been purified using this procedure. A Mono Q Sepharose column was initially used to purify plasmids smaller than 8.0 kb; however, a Hi-Load Q Sepharose column proved more effective with plasmids larger than 8 kb. The loading of plasmids larger than 8 kb on the Mono Q column resulted in a high back pressure and the plasmid DNA could not be eluted from the column. Thus, for routine purification we utilize the Hi-Load Q Sepharose column. Plasmids purified by this method had purity, yield, and transfection efficiency in mammalian cells similar to those of plasmids purified by CsCl density gradient centrifugation.
Chandra
G
G
Molecular Biology Department, Glaxo Inc. Research Institute, Research Triangle Park, North Carolina 27709.
Patel
P
P
Kost
T A
TA
Gray
J G
JG
D002499:Centrifugation, Density Gradient; D002586:Cesium; D002712:Chlorides; D002853:Chromatography, Liquid / Q000379:methods*; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D010957:Plasmids
D002712:Chlorides; D004269:DNA, Bacterial; D002586:Cesium; C028019:cesium chloride