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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 ge tube.
  2. Pellet cells by centrifugation at 250 x g for 5 minutes at 4C.
  3. Decant supernatant and drain any residual supernatant by inverting the tube on a paper towel for 2 minutes.
  4. 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.
  5. After a 5 minute incubation on ice, pellet nuclei by centrifugation at 1100 x g for 12 minutes at 4C.
  6. 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 tube containing the deproteinized DNA. Cap the tube tightly.
  2. Mix by shaking vigorously enough to form a homogeneous suspension. Do not vortex.
  3. Centrifuge at 1500 x g for 5 minutes to isolate the upper, DNA-containing aqueous phase away from the lower, organic solvent phase.
  4. Carefully decant upper phase containing the DNA into a fresh, pre-spun, PLG 15 ml Light tube.
  5. Repeat steps 1 through 3 but this time extracting with 4 ml water-saturated Phenol-Chloroform (PC, 1:1).
  6. Carefully decant upper phase containing the DNA into a clean 15 ml polypropylene centrifuge tube.

    Proceed to Section IV.

IV Precipitation of DNA

At this point, the DNA should have been extracted with both Phenol and Phenol-Chloroform and should be in a clean 15 ml polypropylene screw cap centrifuge tube.

  1. Add 100 l 2 M KCI and mix by gentle inversion.
  2. Overlay DNA solution with 5 ml 95% Ethanol by slowly pipetting the Ethanol down the side of the tube.
  3. Place a Pasteur pipet tip at the interface of the DNA-Ethanol solution and spool the DNA onto the pipet tip by swirling the pipet, keeping the tip at the interface, until the 2 phases are completely mixed.
  4. Place pipet tip with the spooled DNA in 1 ml 70% Ethanol for about 2 minutes.
  5. Remove pipet from the 70% Ethanol and hold upright (tip up) for a few seconds to allow the excess Ethanol to drain away. Do not allow the DNA to dry.
  6. Set pipet tip in a microcentrifuge tube containing 200 l TE (10 mM Tris-Cl, 1 mM EDTA, pH 8.0) and incubate f or 20 minutes at room temperature. The DNA should slide from the pipet into the TE. If necessary, gently remove the DNA from the pipet by scraping the DNA onto the interior of the tube.
  7. Redissolve DNA completely by incubating overnight at 4C. Store DNA at 4C. Do not freeze.
References
  1. Herrmann, B.G. and Frischauf, A.M. (1987) Isolation of genomic DNA. Methods Enzymol. 152:180-183.
  2. Laws, G.M. and S.P. Adams. (1996) Measurement of 8-OHdG in DNA by HPLC/ECD: The importance of DNA purity. BioTechniques 20:36-38.
  3. Murphy, N.R. and Hellwig, R. J. (1996) Improved nucleic acid organic extraction through use of a unique gel barrier matrix. BioTechniques 21:934-939.

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