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DNA Isolation from Fresh or Frozen Solid Tissue Protocol

 

Cell Lysis


    1. Add a piece of frozen ground tissue or fresh tissue (about 50-100 mg or less) to an autoclaved mortar. Add nitrogen immediately and grind hard frozen tissue completely with an autoclaved pestle when nitrogen vaporizes. Repeat until there is a powder-like tissue and transfer to a 15 ml centrifuge tube.  Add 3 ml Cell Lysis Solution.
    2. Incubate lysate at 65°C for 15- 60 minutes. If maximum yield is required, add 15 ml Proteinase K Solution (20 mg/ml) to the lysate, mix by inverting 25 times, and incubate at 55°C for 3 hours to overnight or until tissue particles have dissolved. If possible, invert tube periodically during the incubation.

 

RNase Treatment


    1. Add 15 ml RNase A Solution (4 mg/ml) to the cell lysate.
    2. Mix the sample  by  inverting the tube  25  times  and incubate  at  37°C for 15-60 minutes

 

Protein Precipitation


    1. Cool sample to room temperature.
    2. Add 1 ml Protein Precipitation Solution to the RNase A-treated cell lysate.
    3. Vortex vigorously at high speed for 20 seconds to mix the Protein Precipitation Solution uniformly with the cell lysate.
    4. Centrifuge at 2,000 x g for 10 minutes. The precipitated proteins will form a tight pellet. If the protein pellet is not tight, repeat Step 3 followed by incubation on ice for 5 minutes, then repeat Step 4.

 

DNA Precipitation


    1. Pour the supernatant containing the DNA (leaving behind the precipitated protein pellet) into a clean 15 ml centrifuge tube containing 3 ml 100% Isopropanol (2-propanol).
    2. Mix the sample by gently inverting 50 times.
    3. Centrifuge at 2,000 x g for 3 minutes; the DNA will be visible as a white pellet.
    4. Pour off the supernatant and add 5 ml 70% Ethanol. Invert the tube several times to wash the DNA pellet.
    5. Centrifuge at 2,000 x g for 5 minutes. Carefully pour off the ethanol. The pellet may be loose so pour slowly and watch the pellet.
    6. Invert and drain the tube on clean absorbent paper and allow it to air dry for10-15 minutes.

 

DNA Hydration


    1. Add 150 ml DNA Hydration Solution (150 ml will give a concentration of 500 mg/ml if the total yield is 75 mg DNA).
    2. Re-hydrate DNA by incubating the sample for 1 hour at 65°C and overnight at room temperature. Tap tube periodically to aid in dispersing the DNA.
    3. For storage, sample may be centrifuged briefly and then transferred to a 1.5 ml microfuge tube.
    4. Determine the concentration of the DNA by UV spectrophotometry. Record DNA concentration on sample tube.
    5. Store DNA at 4 °C. For long-term storage, place sample at -20°C or -80°C.

 

Protocol for DNA purification

 

    1. Add equal volume PCI (phenol/chloroform/isoamyl alcohol, 25:24:1) to DNA hydration solution, vortex vigorously, spin 2 mins, remove upper aqueous phase to new tube. Note: Phenol and chloroform are biohazards.  Dispense in a fume hood and wear gloves.
    2. Add 2.5 X volume with 100% ethanol, 10% original volume ammonium acetate (10 M)—can add 1-3 µl glycogen here to preserve low amounts of DNA.  Glycogen makes a nice visible pellet.  Let mixture sit at room temperature for ~2 hours or preferably overnight at –20ºC.
    3. Spin for 20 min at 4 C, decant ethanol (rinse pellet with 75% ETOH if it does not move), air dry.
    4. Carefully re-suspend the pellets in small volumes of hydration buffer and let them dissolve at RT overnight or at 55ºC for 2 hours.
    5. Measure DNA concentration on the Fluorometer.  Ideal DNA concentration is 200-500 µg/ml.