Sample preparation for DNA extraction from ethanol or acetone-fixed tissue

• Spin the contents down and discard the supernatant.
• Add adequate PBS (Phosphate buffer saline) and mix by inverting several times.
• Centrifuge at 600 x g (~3000 rpm) for 5 minutes. Discard the supernatant.
• Repeat the PBS wash steps above.
• Mince or grind the tissue pellet.

To the right is a graph showing DNA extraction from 4 different samples of ethanol-fixed human tissue and measured by a nanospectrophotometer.

Sample preparation for DNA extraction from FFPE tissue

• Slice 2-5 10 µm-thick sections of the tissue using a microtome.
• Remove and discard as much paraffin wax as possible leaving the tissue with less paraffin wax.
• Carry out the xylene method for paraffin wax dissolution.
• Perform the ethanol washes for xylene removal.
• Add 1 ml of PBS (phosphate-buffered saline) and mix by inverting several times.
• Centrifuge at 600 x g (~3000 rpm) for 5 minutes. Discard the supernatant. Repeat the PBS wash step.

To the right is a graph showing DNA extraction from 4 different samples of human FFPE tissue and measured by a nanospectrophotometer.

Paraffin wax removal using mineral oil

• Slice 2-5 10 µm-thick sections of the tissue using a microtome.
• Remove and discard as much paraffin wax as possible leaving the tissue with as little paraffin wax as feasible.
• Take the tissue with little paraffin wax into a 1.5 ml tube.
• Add 200 – 500 µl of mineral oil to the tube and incubate at 70°C for 5 – 10 minutes to accelerate wax dissolution.
  Note: 200 – 300 µl mineral oil is usually sufficient. However, depending on the amount of wax in the tube, more mineral oil may be required.
• Centrifuge at 600 x g (~3000 rpm) for 5 minutes.
• Keep the tube at room temperature (22 – 30°C) for 5 minutes to check for wax resolidification.
  Note: If the wax is not fully dissolved, add more mineral oil and repeat the above three steps until the wax is dissolved.
• Centrifuge at 600 x g (~3000 rpm) for 5 minutes.
• Carefully remove the upper mineral oil layer containing the dissolved paraffin wax and discard it. Take care to remove the upper mineral oil layer as much as possible.