Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli.
Based on the results of a systematic study of factors affecting plasmid yield and purity, a procedure suitable for the rapid screening for and isolation of covalently closed circular DNA from Streptomyces lividans and Escherichia coli was developed. The method consists of lysis of lysozyme-treated bacteria combined with alkaline denaturation of DNA at high temperature. Renaturation of CCC DNA and precipitation of single-stranded DNA together with protein is achieved by the addition of a minimal amount of phenol/chloroform. The screening procedure uses only a single tube and the samples can be analyzed by agarose gel electrophoresis about 30 min after lysis. Removal of phenol and further purification of the plasmid preparation is achieved by consecutive precipitations with isopropanol and spermine, followed by extraction with ethanol, producing samples suitable for restriction endonuclease digestion, ligation, and transformation of S. lividans protoplasts or competent E. coli cells in about 2 h. All steps of the procedure are explained in detail with information about the effects of changing parameters. This should help the experimenter to obtain reproducible results and may be useful if the method has to be adapted to new strains or plasmids.
D002725:Chloroform; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004270:DNA, Circular / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D010636:Phenols; D010957:Plasmids; D012967:Sodium Dodecyl Sulfate; D013302:Streptomyces / Q000235:genetics
D004269:DNA, Bacterial; D004270:DNA, Circular; D010636:Phenols; D012967:Sodium Dodecyl Sulfate; D002725:Chloroform