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Genomic DNA Isolation Protocol(1,2,3)

Note: Genomic DNA is fragile. High molecular weight DNA is sheared easily by mechanical forces. Use suitable large-bore pipet tips or equipment when pipetting genomic DNA. Do not vortex solutions containing genomic DNA.

I Isolation of nuclei

Nuclei from blood

Caution: Wear suitable protective apparel and take appropriate safety measures when working with human blood.

  1. Transfer 5 ml whole blood (containing EDTA as the anti-coagulant) to a 15 ml polypropylene centrifuge tube.
    Note: When working with avian blood, or blood from other species that have nucleated red blood cells, use 0.2 ml blood and 4.8 ml 1x Tris-Buffered Saline (TBS, 50 mM Tris-Cl, 200 mM NaCl, 3 mM KCl, 0.02% Sodium Azide, pH 7.5) instead of 5 ml blood.
  2. Add 5 ml 2x Lysis Buffer (0.65 M Sucrose, 20 mM Tris-Cl, pH 7.8, 10 mM MgCl2, 2% Triton X-100) and mix by gentle inversion.
  3. Incubate solution for 5 minutes on ice.
  4. Pellet nuclei by centrifugation at 1000 x g for 12 minutes at 4C.
  5. Decant supernatant and drain any residual supernatant by inverting the tube on a paper towel for 2 minutes.
    Note: The pelleted nuclei may be stored at 70C for several weeks at this point, however, higher molecular weight DNA is obtained when genomic DNA is isolated from freshly prepared nuclei.

    Proceed to Section II.

Nuclei from tissue culture cells
  1. Suspend cells from one 100 mm plate in the growth medium and transfer them to a 15 ml polypropylene centrifu
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