Isolation of High‐Molecular‐Weight DNA from Mammalian Cells

TLDR

The study presents a preparative method for isolating high‑molecular‑weight DNA from animal cells. The method uses proteinase K with sodium dodecyl sulfate and ethylenediaminetetraacetate to digest proteins. The purified DNA is free of RNA, protein, and degrading enzymes, with a number‑average molecular weight of 190 × 10⁶ (single‑stranded 90 × 10⁶) and a range from 40 × 10⁶ to >500 × 10⁶, indicating intact high‑molecular‑weight molecules.

Abstract

A preparative method for isolating high‐molecular‐weight DNA from animal cells is described. This method is based on the use of proteinase K, a powerful proteolytic enzyme with a broad action spectrum, which is very active in the presence of sodium dodecylsulfate and ethylene‐diamine tetraacetate. The DNA preparation is free of RNA, protein and degrading enzymes. The number‐average molecular weight of the native DNA is 190 × 10 6 , whereas it is 90 × 10 6 for single‐stranded DNA, indicating that the DNA molecules do not contain single‐stranded nicks. The native DNA molecules range in molecular weight from 40 × 10 6 to more than 500 × 10 6 .