Isolation of bacterial plasmids by density gradient centrifugation in cesium trifluoroacetate (CsTFA) without the use of ethidium bromide.
Plasmids extracted from bacterial cells by alkaline extraction can easily be isolated from linear DNA by isopycnic centrifugation in CsTFA. This is a fast and simple method which circumvents the use of the intercalating dye, ethidium bromide, and consequently the problems associated with its removal. The buoyant densities for covalently closed circular DNA and linear DNA in CsTFA are 1.60 g/ml and 1.65 g/ml, respectively. The isolation is achieved regardless of plasmid size and can be accomplished at temperatures of between 4 and 30 degrees C. Plasmid DNA isolated in gradients of CsTFA are of a high purity and have been found to be intact when cleaved with restriction enzymes and ligated with T4 DNA ligase.
D002499:Centrifugation, Density Gradient / Q000379:methods*; D002500:Centrifugation, Isopycnic / Q000379:methods*; D002586:Cesium; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004270:DNA, Circular / Q000302:isolation & purification*; D004926:Escherichia coli / Q000032:analysis; D004996:Ethidium; D005463:Fluoroacetates; D010957:Plasmids; D014269:Trifluoroacetic Acid
D004269:DNA, Bacterial; D004270:DNA, Circular; D005463:Fluoroacetates; D002586:Cesium; D014269:Trifluoroacetic Acid; D004996:Ethidium