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Genomic DNA Isolation Protocol(1,2,3)

ge tube.
  • Pellet cells by centrifugation at 250 x g for 5 minutes at 4C.
  • Decant supernatant and drain any residual supernatant by inverting the tube on a paper towel for 2 minutes.
  • Suspend cells in 1 ml 1x TBS by repeated up and down pipetting, then add 4 ml 1x TBS and 5 ml 2x Lysis Buffer to the cell suspension and mix by inversion.
  • After a 5 minute incubation on ice, pellet nuclei by centrifugation at 1100 x g for 12 minutes at 4C.
  • Decant supernatant and drain any residual supernatant by inverting the tube on a paper towel for 2 minutes.
    Note: The nuclei may be stored at 70C for several weeks at this point, however, higher molecular weight DNA is obtained from freshly prepared nuclei.

    Proceed to Section II.

  • II Lysis of nuclei and deproteinization of DNA
    1. Suspend pelleted nuclei in 2 ml Saline/EDTA Solution (75 mM NaCl, 24 mM EDTA) by repeated up and down pipetting.
    2. Carefully transfer suspended nuclei to a pre-spun (1500 x g for 23 minutes) PLG 15 ml Light tube.
    3. Add 50 L 20 mg/ml Proteinase K (in 10 mM Tris-Cl, pH 8.0, 1.0 mM CaCl2, 30% Glycerol) and 200 l 10% SDS to the suspended nuclei and mix by gentle inversion.
    4. Incubate for 2 hours at 37C with occasional gentle mixing. Note: This incubation may be extended to 1618 hours if that is more convenient.

      Proceed to Section III.

    III Extraction of protein
    1. Add 4 ml water-saturated Phenol to the PLG 15 ml Light
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