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Genomic DNA Purification for Paraffin-embeded Tissue

  1. Place no more than 30 mg of tissue (~2 mm3 ) in a clean 2 ml microtube.

  2. Extract the sample with 1 ml xylene to remove the paraffin. Mix thoroughly by vortexing.

  3. Centrifuge the tube at 10,000 x g for 10 min at room temperature. Discard the supernatant without disturbing the pellet.

  4. Rinse the pellet with 1 ml absolute ethanol (96-100%) to remove any traces of xylene. Centrifuge at 10,000 x g for 5 min at room temperature. Discard the ethanol without disturbing the tissue pellet. 

  5. Repeat the ethanol rinse.

  6. Air dry the tissue pellet at 37°C for 15 min.

  7. Add 200 μl Buffer TL to the tissue.

  8. Add 25 μl Proteinase K (20 mg/ml). Slightly vortex to mix well, and incubate in a shaking water bath (55°C) for 1-4 hours or until lysis is complete. If no shaking water-bath is available, vortex the sample every 20-30 min.  Incomplete lysis may block column flow and significantly reduce DNA yields.

  9. OPTIONAL: Paraffin-embedded tissue have low levels of RNA which will be co-purified with DNA using this kit. While it will not interfere with PCR, the RNA may be removed at this point. To do so, add 4 μl RNase A (100 mg/ml) and incubate at room temperature for 2 min. Proceed with the following protocol.

  10. Centrifuge at 12,000 x g for 5 min to pellet insoluble tissue debris and hair. CAREFULLY aspirate the supernatant, and transfer to a sterile 1.5 ml microtube, while leaving behind any insoluble pellet.

  11. Add one volume Buffer BL followed by one volume absolute ethanol. Vortex thoroughly to mix. Alternatively, user may add 2 volumes of premixed BL/ethanol mixture to the sample. Thorough mixing is essential at this point.

  12. Transfer the entire sample into the column including any wispy precipitate that may have formed. Centrifuge at 10,000x g for 30 - 60 s to bind DNA. Discard flow-through.

  13. Place the Mini Column into a new collection tube. Add 500 μl Buffer KB. Centrifuge at 10,000 x g for 1 min. Discard collection tube and flow-through liquid.

  14. Place the Mini Column into a new collection tube. Add 700 μl DNA Wash Buffer diluted with absolute ethanol. Centrifuge as above and discard flow-through liquid. Ensure that ethanol has been added to DNA Wash Buffer. If refrigerated, DNA Wash Buffer must be brought to room temperature before use.

  15. Using the same 2 ml collection tube, repeat Step 8.

  16. Using the same 2 ml collection tube, centrifuge the column with the lid open at a maximum speed (13,000 x g ) for 2 min to remove the residual ethanol.  This step is crucial for ensuring optimal elution in the following step.

  17. Place the column into a sterile 1.5 ml microtube. Add 100-200 μl Elution Buffer (preheated to 70°C, 10 mM Tris-HCl, pH 8.5). Allow to sit at room temperature for 3 min. Centrifuge at 13,000 x g for 1 min.

NOTES:

  • For the eluting of DNA, we recommend using 50-100 μl of Elution Buffer warmed to 70°C. Yields will depend on size and age of sample. Certain samples may require prolonged lysis with Buffer BL.
  • Tissue fixed with paraformaldehyde will yield degraded DNA or RNA. The extent of degradation depends on type of fixative used, but the size of DNA obtained is usually less than 500 bp. Degradation is not caused by the EZgene™ Tissue DNA Protocol. For PCR detection of segments smaller than 500 bp, satisfactory results can be obtained.
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