Rapid purification of Ti plasmids from Agrobacterium by ethidium bromide treatment and phenol extraction.
An efficient method is described for the purification of Ti plasmid DNA from Agrobacterium. The procedure is based on the relative binding capacity of ethidium bromide to supercoiled plasmid DNA and linear DNA and on the high solubility of ethidium bromide in phenol. Following treatment with ethidium bromide, more than 87% of linear chromosomal DNA and most of the RNA was present in the phenol phase, while 91% of Ti plasmid DNA was recovered from the aqueous phase. The Ti plasmid DNA was sufficiently pure for restriction endonuclease analysis and cloning. The procedure is simple, fast and provides eight times higher yield than the standard isopycnic ultracentrifugation method.
Zhang
L
L
Waite Agricultural Research Institute, University of Adelaide, Glen Osmond, SA, Australia.
Kerr
A
A
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D002499:Centrifugation, Density Gradient; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004278:DNA, Superhelical / Q000302:isolation & purification; D004996:Ethidium; D010636:Phenols; D010957:Plasmids / Q000302:isolation & purification*; D012150:Polymorphism, Restriction Fragment Length; D012995:Solubility
D004269:DNA, Bacterial; D004278:DNA, Superhelical; D010636:Phenols; D004996:Ethidium