DNA Isolation from Fresh or Frozen Solid Tissue Protocol
- 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.
- 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.
- Add 15 ml RNase A Solution (4 mg/ml) to the cell lysate.
- Mix the sample by inverting the tube 25 times and incubate at 37°C for 15-60 minutes
- Cool sample to room temperature.
- Add 1 ml Protein Precipitation Solution to the RNase A-treated cell lysate.
- Vortex vigorously at high speed for 20 seconds to mix the Protein Precipitation Solution uniformly with the cell lysate.
- 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.
- 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).
- Mix the sample by gently inverting 50 times.
- Centrifuge at 2,000 x g for 3 minutes; the DNA will be visible as a white pellet.
- Pour off the supernatant and add 5 ml 70% Ethanol. Invert the tube several times to wash the DNA pellet.
- 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.
- Invert and drain the tube on clean absorbent paper and allow it to air dry for10-15 minutes.
- Add 150 ml DNA Hydration Solution (150 ml will give a concentration of 500 mg/ml if the total yield is 75 mg DNA).
- 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.
- For storage, sample may be centrifuged briefly and then transferred to a 1.5 ml microfuge tube.
- Determine the concentration of the DNA by UV spectrophotometry. Record DNA concentration on sample tube.
- Store DNA at 4 °C. For long-term storage, place sample at -20°C or -80°C.
Protocol for DNA purification
- 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.
- 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.
- Spin for 20 min at 4 C, decant ethanol (rinse pellet with 75% ETOH if it does not move), air dry.
- Carefully re-suspend the pellets in small volumes of hydration buffer and let them dissolve at RT overnight or at 55ºC for 2 hours.
- Measure DNA concentration on the Fluorometer. Ideal DNA concentration is 200-500 µg/ml.
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