DNA Isolation from Paraffin Embedded Tissue Samples

The procedure is developed for use at the Department of Clinical Research, Hvidovre Hospital.
The aim of this instruction is to insure the correct isolation of DNA from paraffin embedded tissue.

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Materials:

• Proteinase K 20 mg/ml stock solution (in water). Store at  –20 degrees. May be frozen and thawed several times (100mg, in 5 ml sterile water. Aliqoute 310ul) 
• Digestion buffer; 5 ml Nuclei Lysis solution (from DNA Wizard Extraction kit, Promega) mixed with 1,2 ml EDTA (pH 8,0)
• 7,5 M ammonium acetat
• PCI (phenol/chloroform/isoamyl alcohol) Ultra pure (fra Invitrogen)
• Xylene min 98% (Group C, Fraction 03.11)
• 99,9% Ethanol
• Glycogen
• ddH₂O

Procedure:

1. Put 3 x 10 µm sections of formalin fixed paraffin samples (or 1-2 x 1mm cores) into Eppendorf tubes
2. Add 1 mL xylene, mix on nutator for 15 minutes. Spin 2 minutes in Eppendorf ultramicrofuge, and discard supernatant. (Perform all the spinding at 14.000 rcf)3.   Repeat Step 2 two more times, removing as much xylene as possible. First remove most of the xylene
3. Briefly spin, take off more, spin again, and take off the final amount
4. Add 1 ml 99,9% Ethanol and mix on nutator at room temp for 15 minutes. Spin down
5. Remove Ethanol. Allow pellet to air dry
6. Add up to 500 µL of digestion buffer
7. Add 25 µL fresh Proteinase K
8. Incubate overnight at 55°C (Themormixer) 550 rpm
9. Repeat steps 7 and 8 for two more days
10. Add equal volume of PCI (575µL) to the Proteinase K digested aqueous solution, shake gently and incubate for 10 minutes at room temperature. Spin 2 minutes and remove upper aqueous phase, ~500 µL, to a new tube
11. Add another 500µL PCI. Flip the tube a few times (shake gently), and incubate at RT for 10 min. Centrifuge for 2 min, and transfer the upper aqueous phase to a new 2 ml tube
12. Add 300 µL ammonium acetate (7.5M), 1 mL ice-cold Ethanol and 5 µL glycogen. Leave at -20 °C for 1-2 hours
13. Spin for 45 minutes at 4°C, decant ethanol, air dry
14. Carefully re-suspend the pellets in small volumes of ddH2O (approximately 20-100 µL) and let dissolve at room temperature overnight
15. Measure DNA concentration using the Fluorometer. Ideal DNA concentration is from 200-600 µg/mL
16. Store DNA at 4 degrees

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