PROTOCOL TO EXTRACT DNA FROM PA

PROTOCOL TO EXTRACT DNA FROM PARAFFIN BLOCKS

1.Cut 10-20X10μm sections of formalin fixed paraffin samples into eppendorf tubes.

2.Add 1 ml xylene, mix, incubate at 55℃ for 15mins. Release pressure, spin down for 2minutes in eppendorf ultramicrofuge, pipet off & discard supernatent.

3.Repeat Step 2.

4.Add 100% ethanol, incubate for 15mins. Spin down.

5.Remove ethanol, Repeat Step 4, allow pellet to air dry.

6.Add up to 1ml of in to a final concentration of 0.3-0.5mg/ml (less buffer for smaller tumors).

7.Incubate overnight, shaking, at 55℃.

8.Add fresh concentrated PK (same amount as day 1 from 20mg/ml stock).

9.Incubate overnight at 55℃.

10.Repeat step 8 & 9.

11.Add equal volume to the PK digested aqueous solution, spin 2mins, remove upper aqueoues phase to new tube. (repeat PCI extraction if necessary -- it usually is).

12.Take 330μl aqueous phase per eppendorf tube, add 1/2 original volume (165μl) ammonium acetate (7.5M) [can add 1-3μl Glycogen here to preserve low amounts of DNA. Glycogen makes a nice visible pellet] and 2-2.5X volume with 100% ethanol. Let sit at RT for 2 hours or overnight at -20℃.(better O.N.).

13.Spin for 20minutes at 4℃, decant ethanol (rinse pellet with etoh if it doesn't move), blot dry, air dry.

14.Carefully resuspend the pellets in small volumes of TE buffer (15-20μl) and let dissolve at RT overnight (or 55℃ for 2hours). Combine tubes from the same sample.

15.Measure DNA concentration on the Fluorometer. Ideal DNA concentration is from 200-500μg/ml.

16.Denature the DNA at 70℃ before running 0.2μg on 1% gel to check the size. Size should range from 100bp-3Kb.

Solutions

Digestion buffer : 100mM NaCl/10mM Tris-HCl, pH 8.0 and 25mM EDTA, pH 8.0/0.5% SDS. Store at RT.

Proteinase K: Stored as 20mg/ml aliquots at -20℃; Can be refrozen a few times.

Use Proteinase K at 0.3-0.5mg/ml in digestion buffer

PCI : phenol/chloroform/isoamyl alcohol (from Ameresco)