Preparation of transforming deoxyribonucleic acid by phenol treatment

Abstract

Several procedures employing chloroform and phenol for extraction and deproteinization have been explored for the preparation of highly purified transforming DNA from Bacillus subtilis. The recovery, purity, transforming activity and the sedimentation coefficients of the preparations have been compared. The following procedure has been found to give the best results: lysis of cells with lysozyme (EC 3.2.1.17), followed by freezing and thawing, extraction of nucleic acids with a mixture of phenol and pH-9 buffer in the presence of sodium dodecyl sulfate, digestion of RNA with a mixture of pancreatic RNAase (EC 2.7.7.16) and RNAase T1 from Taka-diastase (EC 3.1.4.8), followed by treatment with phenol. When the digestion is carried out with pancreatic RNAase alone, it is followed by precipitation with isopropapol in order to eliminate RNA. The final DNA preparation has high transforming activity, especially for the joint transformation of linked markers. © 1963.