Method for the preparation of plasmid DNA suitable for physicochemical measurements.
A method has been developed for the isolation of plasmid DNA suitable for physical and physicochemical measurements. The procedure is based on the deproteinization of the cleared lysate of bacterial cells (after amplification of plasmids by chloramphenicol) by phenol at pH 8.0 and subsequent removal of chromosomal DNA by means of phenol at pH 4.0 and separation of RNA on a hydroxyapatite column at higher temperature. ColE1 DNA sample was compared with samples of the same DNA prepared by three thus far used methods. Samples obtained by means of the latter methods were contaminated with chromosomal DNA, RNA, or ethidium bromide. The presence of ethidium bromide in the DNA sample was a factor interfering in the electrochemical analysis, chromosomal DNA and RNA were disturbing in the use of other methods. DNA separated by the method devised by us was free of any detectable contaminants and fulfilled the high requirements for sample purity of differential pulse polarography. Measurements performed by means of differential pulse polarography showed that the content of single-stranded segments in superhelical ColE1 DNA is less than 0.15% (i.e. less than 20 bases per molecule). This is in keeping with the notion that a cruciform is formed in this DNA (as a result of tension due to supercoiling) in the region of inverted repeat sequence, containing only 5 bases in the single-stranded loop region.
D000818:Animals; D002417:Cattle; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004277:DNA, Single-Stranded / Q000032:analysis; D004278:DNA, Superhelical / Q000302:isolation & purification; D004996:Ethidium; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011048:Polarography / Q000379:methods; D013950:Thymus Gland / Q000032:analysis
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D004996:Ethidium