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Genomic DNA Isolation Protocol(1,2,3)

tube containing the deproteinized DNA. Cap the tube tightly.
  • Mix by shaking vigorously enough to form a homogeneous suspension. Do not vortex.
  • Centrifuge at 1500 x g for 5 minutes to isolate the upper, DNA-containing aqueous phase away from the lower, organic solvent phase.
  • Carefully decant upper phase containing the DNA into a fresh, pre-spun, PLG 15 ml Light tube.
  • Repeat steps 1 through 3 but this time extracting with 4 ml water-saturated Phenol-Chloroform (PC, 1:1).
  • 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
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