Isolation of High-Molecular-Weight DNA from Mammalian Cells

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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 × 106, whereas it is 90 × 106 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 × 106 to more than 500 × 106.

Abbreviation
SV40

Simian virus 40

Enzymes
 

Pancreatic ribonuclease (EC 2.7.7.16)

 

T1 ribonuclease (EC 2.7.7.26)

Ancillary