Purification of RNA-free plasmid DNA using alkaline extraction followed by Ultrogel A2 column chromatography.
A procedure for extracting RNA-free plasmid DNA from bacterial cells is described. The method is simple and rapid enough to obtain pure plasmid DNA in 8 to 10 h after plasmid amplification. The protocol uses the alkaline extraction procedure described by Birnboim and Doly (1979, Nucl. Acid Res. 7, 1513-1523). Plasmid DNA is then separated from high-molecular-weight RNA by ammonium acetate precipitation and from low-molecular-weight RNA contaminants by Ultrogel A2 column chromatography. The plasmid DNA obtained by this inexpensive technique is sufficiently pure to be used for restriction endonuclease analysis, 5'-end labeling, S1 mapping, DNA sequencing, and colony hydridization.
Micard
D
D
Sobrier
M L
ML
Couderc
J L
JL
Dastugue
B
B
D002845:Chromatography / Q000295:instrumentation; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010957:Plasmids; D012329:RNA, Bacterial / Q000302:isolation & purification
D004269:DNA, Bacterial; D012329:RNA, Bacterial