This paper describes a method for isolating high-molecular-weight DNA from mammalian cells using proteinase K, a powerful proteolytic enzyme. The method is based on the use of sodium dodecylsulfate and ethylenediamine tetraacetate to enhance the activity of proteinase K. The resulting DNA preparation is free of RNA, protein, and degrading enzymes, with a number-average molecular weight of 200×10^6, and no evidence of single-stranded nicks. The authors demonstrate that their method yields a high-purity DNA suitable for studies of transcription in vitro, as it is larger than the size of an average transcription unit and free of single-stranded nicks. The method is also applicable to the preparation of DNA from other sources, such as calf thymus and prokaryotes, and can be used for studying DNA structure and replication by electron microscopy.This paper describes a method for isolating high-molecular-weight DNA from mammalian cells using proteinase K, a powerful proteolytic enzyme. The method is based on the use of sodium dodecylsulfate and ethylenediamine tetraacetate to enhance the activity of proteinase K. The resulting DNA preparation is free of RNA, protein, and degrading enzymes, with a number-average molecular weight of 200×10^6, and no evidence of single-stranded nicks. The authors demonstrate that their method yields a high-purity DNA suitable for studies of transcription in vitro, as it is larger than the size of an average transcription unit and free of single-stranded nicks. The method is also applicable to the preparation of DNA from other sources, such as calf thymus and prokaryotes, and can be used for studying DNA structure and replication by electron microscopy.