A method is described for isolating high-molecular-weight DNA from mammalian cells. The method uses proteinase K, a powerful proteolytic enzyme, in the presence of sodium dodecylsulfate and ethylenediamine tetraacetate. The resulting DNA preparation is free of RNA, protein, and degrading enzymes. The native DNA has a number-average molecular weight of 200×10⁶, with no evidence of single-stranded nicks. The DNA molecules range in molecular weight from 40×10⁶ to over 500×10⁶. The method is effective in producing DNA with high molecular weight and minimal degradation, suitable for studies of transcription in vitro. The DNA is purified through a series of phenol extractions and dialysis steps, and its purity is confirmed by various assays. The sedimentation coefficient and molecular weight of the DNA are calculated using standard methods. The DNA is also analyzed by electron microscopy, showing long, well-extended molecules with no single-stranded regions. The results indicate that the DNA molecules are intact and suitable for studying transcription and DNA structure. The method is applicable not only to mammalian cells but also to other organisms, and it can be used for isolating intact RNA molecules. The study highlights the importance of using high-quality DNA for accurate transcription studies and provides a reliable method for obtaining such DNA.A method is described for isolating high-molecular-weight DNA from mammalian cells. The method uses proteinase K, a powerful proteolytic enzyme, in the presence of sodium dodecylsulfate and ethylenediamine tetraacetate. The resulting DNA preparation is free of RNA, protein, and degrading enzymes. The native DNA has a number-average molecular weight of 200×10⁶, with no evidence of single-stranded nicks. The DNA molecules range in molecular weight from 40×10⁶ to over 500×10⁶. The method is effective in producing DNA with high molecular weight and minimal degradation, suitable for studies of transcription in vitro. The DNA is purified through a series of phenol extractions and dialysis steps, and its purity is confirmed by various assays. The sedimentation coefficient and molecular weight of the DNA are calculated using standard methods. The DNA is also analyzed by electron microscopy, showing long, well-extended molecules with no single-stranded regions. The results indicate that the DNA molecules are intact and suitable for studying transcription and DNA structure. The method is applicable not only to mammalian cells but also to other organisms, and it can be used for isolating intact RNA molecules. The study highlights the importance of using high-quality DNA for accurate transcription studies and provides a reliable method for obtaining such DNA.