Rapid step-gradient purification of mitochondrial DNA.
A convenient modification of the step gradient (CsCl/ethidium bomide) procedure is described. This rapid method allows isolation of covalently closed circular DNA separated from contaminating proteins, RNA and chromosomal DNA in ca. 5 h. Large scale preparations can be performed for circular DNA from eukaryotic organelles (mitochondria). The protocol uses organelle pelleting/NaCl-sarcosyl incubation steps for mitochondria followed by a CsCl step gradient and exhibits yields equal to the conventional procedures. It results in DNA sufficiently pure to be used for restriction endonuclease analysis, subcloning, 5'-end labeling, gel retention assays, and various types of hybridization.
Welter
C
C
Institute of Human Genetics, University of the Saar, Homburg, FRG.
Meese
E
E
Blin
N
N
D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient / Q000379:methods*; D002586:Cesium; D002712:Chlorides; D004270:DNA, Circular / Q000302:isolation & purification; D004272:DNA, Mitochondrial / Q000302:isolation & purification*; D004996:Ethidium; D006801:Humans; D010957:Plasmids
D002712:Chlorides; D004270:DNA, Circular; D004272:DNA, Mitochondrial; D002586:Cesium; D004996:Ethidium; C028019:cesium chloride