Patent ID: 8546134

Claim:
A method for isolation of nuclei having their nuclear outer membranes cleanly removed and their nuclear inner membranes substantially intact, comprising: (a) providing a sample of mammalian, avian or amphibian cells; (b) mechanically disrupting the cells in a mildly hypertonic buffer comprising about 250 mM sucrose to generate a disrupted cell sample; (c) centrifuging the disrupted cell sample at 500-1000×g in a microcentrifuge for 5-15 minutes to pellet insoluble materials forming a first supernatant and a first crude nuclei pellet; (d) separating the first supernatant from the first crude nuclei pellet; (e) resuspending the pellet in a highly hypertonic buffer comprising about 2 M sucrose; (f) centrifuging the resuspended pellet of (e) at 10,000-20,000×g in a microcentrifuge for 10-30minutes to generate a second nuclei pellet and a second supernatant; (g) separating the second supernatant and isolated pellet of (f), wherein the isolated nuclei pellet contains purified nuclei which have their nuclear outer membranes cleanly removed and their nuclear inner membranes substantially intact.