DNA extraction - Salting Out protocol

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Solutions/reagents:

  • <a name="Digestion Buffer">Digestion Buffer
    <tab>
    (10mM NaCl, 10mM TRIS (pH 8.0), 10mM EDTA (pH 8.0), 0.5% SDS)
    </a>
  • <a name="Proteinase K">Proteinase K
    <tab>
    (20mg/ml)
    </a>
  • <a name="Sodium Acetate pH 5.2">Sodium Acetate pH 5.2
    <tab>
    (3M)
    </a>
  • ice-cold 98% ethanol
  • ice-cold 70% ethanol
  • 1X TE
  • water
  • tissue

Equipment:

  • Incubator
  • Centrifuge
  • Sterile 1.5-ml microcentrifuge tubes

Steps:

  1. Tissue Digestion

    1. Measure out <a href="#Digestion Buffer" >Digestion Buffer</a> into sterile 1.5-ml microcentrifuge tube (2).
      Add 0.005 volume <a href="#Proteinase K" >Proteinase K</a>.
      That is, for each ml of Digestion Buffer, add 5 µl of ProteinaseK.

    2. Homogenize tissue in solution.

    3. Incubate at 55°C for 1 - 12 hrs(overnight).

    4. Vortex the mixture for a few secs.
      Centrifuge at maximum speed for 2 mins at 4°C and aspirate out the top layer.
      Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (3).
      Discard bottom layer.

  2. Precipitation of Protein and Cell Debris

    1. Add 0.1 volume <a href="#Sodium Acetate pH 5.2" >Sodium Acetate pH 5.2</a> to sterile 1.5-ml microcentrifuge tube (3).

    2. Close the tube tightly and gently mix the contents by inverting the tube.
      Incubate at -20°C for 15 mins.
      Centrifuge at maximum speed for 20 mins at 4°C and aspirate out the top layer.
      Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (4).
      Discard bottom layer.
      Be careful not to transfer any of the white solid (cell debris and SDS) into the fresh tube.

  3. Precipitation of Nucleic Acids

    1. Add 2 volumes ice-cold 98% ethanol to sterile 1.5-ml microcentrifuge tube (4).

    2. Close the tube tightly and gently mix the contents by inverting the tube.
      Incubate at -20°C for 15 mins.

    3. Centrifuge at maximum speed for 20 mins at 4°C, gently aspirate out the supernatant and discard it.

    4. Add 1 ml of ice-cold 98% ethanol.
      Vortex the mixture for a few secs.
      Centrifuge at maximum speed for 5 mins at 4°C, gently aspirate out the supernatant and discard it.
      Add 1 ml of ice-cold 70% ethanol.
      Vortex the mixture for a few secs.
      Centrifuge at maximum speed for 5 mins at 4°C, gently aspirate out the supernatant and discard it.

    4. (Optional)
    Add 1 ml of ice-cold 70% ethanol.
    Vortex the mixture for a few secs.
    Centrifuge at maximum speed for 5 mins at 4°C, gently aspirate out the supernatant and discard it.

  4. Dry the pellet in air.

    Option 1: Add 10 µl of 1X TE.
    (or)
    Option 2: Add 10 µl of water.

    Resuspend pellet by vortexing/by shaking vigorously.
    Ensure to dry the pelletted DNA completely before attempting to resuspend.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 13 hrs, 27 mins

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