| 1. | 2 X 15 minute wash in xylene or equivalent completely immersing the section or slide in the wash agent.
( 100% Acetone Wash @ 2 x 5 minutes is required for all lipid-rich tissue, i.e brain tissue, etc.) |
| 2. | Wet slide or tissue by sequential washes in:
- 100% Ethanol 2x5 min.
- 70% Ethanol 2x5 min.
- 30% Ethanol 2x5 min.
- 100% DI water 2x5 min.
|
| 3. | Wash slide or section:
- 1xTE 1x5 min.
- 100% DI water 1x5 min.
|
| 4. | Combine tissue and 20µl of GeneReleaser® a into 1.5ml tube. If tissue is slide mounted then use approximately a 1mm2 to 9mm2 tissue scraping into tube. |
| 5. | Using a disposable pestle grind the tissue in the tube about 10-20 thrust with the pestle. Alternatively, 10 units of Proteinase K can be added and the tissue digested for 30 minutes at 55°C in a capped tube. |
| 6. | Transfer as much of the contents as possible of the 1.5ml tube containing the GR treated tissue to a 0.5ml PCR tube. |
| 7. | Overlay with mineral oil and treat using the microwave protocol. |
| 8. | Add 25µl of 1XTE to the treated tissue in the 0.5ml tube. |
| 9. | Centrifuge for 5 minutes at 5,000 x g |
| 10. | Transfer the aqueous phase to a fresh tube. |
| 11. | Use 1-10µl of the aqueous phase from step 10 as a source of DNA for PCR. |
| Note: the volume required depends on the amount of tissue, the type of tissue, fixation method and age of specimen. |
