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Genomic DNA purification -Animal Tissue or Mouse Tail

Protocol for Tissues

Tissue preparation and lysis:

  • Using Homogenizer: Using a blade to slice tissue, weigh out 10-30 mg of animal tissue and transfer it to a microcentrifuge tube, add 500 μl Lysis Buffer (with β-mercaptoethanol added), homogenize the tissue according to the manufacturer’s instruction

  • Using liquid N2: immediately place the weighted tissue in liquid nitrogen, and grind thoroughly with a mortar and pestle. Pour  tissue powder and liquid nitrogen into a microcentrifuge tube. Allow the liquid nitrogen to evaporate, but don’t let the tissue to thaw. Add 500 μl Lysis Buffer, homogenize through a 20-gauge needle fitted to a syringe

  • Proteinase K digested tissue lysate (up to 125 μl), add 500 μl Lysis Buffer and mix well.

  1. Centrifuge the homogenized tissue lysate at 11,000 rpm for 5 min. Transfer the supernatant to a gDNA binding column. Don’t disturb the pellet during transfer. Centrifuge at 11,000 rpm for 30-60 sec. Discard the flowthrough.

  2. Add 400 μl Lysis Buffer to the column, centrifuge at 11,000 rpm for 30-60 sec. Discard the flowthrough.

  3. Add 700 μl DNA Wash Buffer to the column, centrifuge at 11,000 rpm for 30-60 sec. Discard the flowthrough. Make sure that ethanol is added to washing buffer as instructed before use.

  4. Centrifuge additional 30-60 sec at 11,000 rpm. It is critical to remove residual ethanol for optimal elution. Residual ethanol may interfere subsequent applications.

  5. Place the DNA column in a clean 1.5 ml microcentrifuge tube. Add 50-100 µl pre-heated (70oC waterbath) DNA Elution Buffer or Nuclease-Free water to the center of the column, incubate for 1 min, centrifuge at 11,000 rpm  for 30-60 sec to elute the DNA.

The purified DNA is stable in elution buffer or water if stored at -20°C or below.

Protocol for Cells

Cell preparation and lysis:

  • Adherent cells: scrape 1-5x10cultured cells from the culture dish or plate, pellet cells by centrifugation. Completely remove the supernatant by aspiration.

  • Suspension cells: pellet 1-5x10cells by centrifugation. Completely remove the supernatant by aspiration.

  1. Add 500 μl Lysis Buffer, vortex to resuspend the cell pellet. Homogenize the lysate by passing it through a 20-gauge needle fitted to a DNase free syringe for at least 5 times.

  2. Transfer the homogenized lysate to a gDNA binding column. Centrifuge at 11,000 rpm for 30-60 sec. Discard the flowthrough.

  3. Add 400 μl Lysis Buffer to the column, centrifuge at 11,000 rpm for 30-60 sec. Discard the flowthrough.

  4. Add 700 μl DNA Wash Buffer to the column, centrifuge at 11,000 rpm for 30-60 sec. Discard the flowthrough. Make sure that ethanol is added to washing buffer as instructed before use.

  5. Centrifuge additional 30-60 sec at 11,000 rpm. It is critical to remove residual ethanol for optimal elution. Residual ethanol may interfere subsequent applications.

  6. Place the DNA column in a clean 1.5 ml microcentrifuge tube. Add 50-100 µl pre-heated (70oC waterbath) DNA Elution Buffer or Nuclease-Free water to the center of the column, incubate for 1 min, centrifuge at 11,000 rpm for 30-60 sec to elute the DNA.

The purified DNA is stable in elution buffer or water if stored at -20°C or below.

 

 

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