PROTOCOL TO EXTRACT DNA FROM PARAFFIN BLOCKS

Cut 10-20X 10 um sections of formalin fixed paraffin samples into eppendorf tubes. Add 1 ml xylene, mix, incubate at 55 C for 15 mins. Release pressure, spin down for 2 minutes in eppendorf ultramicrofuge, pipet off & discard supernatent. Repeat Step 2. Add 100% ethanol, incubate for 15 mins. Spin down. Remove ethanol, Repeat Step 4, allow pellet to air dry. Add up to 1 ml of Proteinase K in digestion buffer to a final concentration of 0.3-0.5 mg/ml (less buffer for smaller tumors). Incubate overnight, shaking, at 55 C. Add fresh concentrated PK (same amount as day 1 from 20 mg/ml stock). Incubate overnight at 55 C. Repeat step 8 & 9. Add equal volume PCI to the PK digested aqueous solution, spin 2 mins, remove upper aqueoues phase to new tube. (repeat PCI extraction if necessary -- it usually is). Take 330 ul aqueous phase per eppendorf tube, add 1/2 original volume (165ul) ammonium acetate (7.5M) [can add 1-3 ul 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 C.(better O.N.). Spin for 20 minutes at 4 C, decant ethanol (rinse pellet with etoh if it doesn't move), blot dry, air dry. Carefully resuspend the pellets in small volumes of TE buffer (~15-20 ul) and let dissolve at RT overnight (or 55 C for 2 hours). Combine tubes from the same sample. Measure DNA concentration on the Fluorometer. Ideal DNA concentration is from 200-500 ug/ml. Denature the DNA at 70 C before running 0.2 ug on 1% gel to check the size. Size should range from 100bp-3Kb.

  Solutions

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

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

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

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

Warning!!!!!!!! xylene and ethanol are flammable and heating flammable solvents is dangerous. The flashpoint of xylene is 27oC. Ethanol has a flashpoint of 12 oC . (Flashpoint is the minimum temperature at which a liquid gives off a vapor in sufficient concentration to ignite). Because of the inherent dangers of heating flammable solvents: 1.) Always conduct experiments in a chemical fume hood 2.) Ensure that the hood is free from all potential sources of ignition including electrical outlets, power strips, and electrical equipment, flames ect. 3.) Remove all other flammable solvents and oxidizers from the hood before beginning. 4.) Wear a lab coat, chemically resistant gloves, and a face shield to protect against splashing. 5.) Heating solvents in closed tubes can lead to pressure build up inside the tubes causing the cap to pop open and potential splashing. The cap should be opened to relieve pressure before handling or kept open during the 15 minute incubation.