DNA Extraction for Formalin Specimen

Place the tissue in a 1.5 ml or 2 ml microcentrifuge tube. Add 300 µl Deparaffinization Solution , vortex vigorously for 10 s , and centrifuge briefly to bring the sample to the bottom of the tube.

Note: For specimens stored only in alcohol (=not fixed in paraffin), "deparaffinization step" is not required. In this case, instead of 300 µl Deparaffinization Solution, add 300 µl ATL, 300 µl AL , and 300 μl ethanol (96–100%) , and proceed to the next step 3 , without step 2 (incubation).

Incubate at 56°C for 3 min , then allow to cool to room temperature .

Add 25 µl Buffer FTB , 55 µl RNase-free Water , and 20 µl Proteinase K . Mix by vortexing. Briefly centrifuge the tube to spin down any tissue that sticks to the tube wall or under the cap of the tube after vortexing.

Incubate for 2 h at 56°C and 1000 rpm.

Note: In general, more than one hour is required, and the results were most successful when the process was conducted for more than 2 hours. For relatively older samples, it can be proceed "overnight".

Incubate for 1 h at 90°C without shaking.

Carefully remove and discard the upper blue phase . Keep the lower aqueous lysate, add 150 µl RNase-free Water , then vortex.

Note: If the deparaffinization process (step 1 and 2) is omitted, remove the 900 µl of the upper lysate .

Add 2 μl RNase A , vortex, and incubate for 2 min at room temperature on the bench.

Add 20 µl Proteinase K , vortex, and incubate for 15 min at 65°C and 450 rpm .

Add 250 μl Buffer AL and 250 μl ethanol (96–100%) to each sample and mix thoroughly by vortexing

Transfer 450 µl lysate to the QIAamp UCP MinElute column (in a 2 ml collection tube), and centrifuge at 15,000 rpm for 30 s .

Note: Maximum speed is recommended.

Transfer the residual lysate to the same QIAamp UCP MinElute column, and centrifuge at 15,000 rpm for 1 min . Discard the flow-through from step 10 and 11 and reuse the collection tube.

Note: Maximum speed is recommended.

Add 500 μl Buffer AW1 to each spin column, and centrifuge at 15,000 rpm for 30 s . Discard the flow-through and reuse the collection tube.

Note: Maximum speed is recommended.

Add 500 μl Buffer AW2 to each spin column, and centrifuge at 15,000 rpm for 30 s . Discard the flow-through and reuse the collection tube.

Note: Maximum speed is recommended.

Add 250 μl ethanol (96–100%) to the spin column, and centrifuge at 15,000 rpm for 30 s . Discard the flow-through and collection tube. Place the spin column into a new 2 ml collection tube and centrifuge for 3 min at full speed to remove any residual liquid to dry the membrane.

Note: Maximum speed is recommended.

Place the QIAamp UCP MinElute column into a clean 1.5 ml microcentrifuge tube, and discard the collection tube containing the flow-through. Open the lid of the QIAamp MinElute column and apply 20 μl Buffer ATE to the center of the membrane.

Close the lid and incubate at room temperature for 1 min , then centrifuge at full speed for 1 min to elute the DNA. Step 15 is repeated twice more so that final volume is 60 μl (20 μl+20 μl+20 μl) .

Note: The final volume can be up to 100 μl, but the sequence result in the case of 60 μl was the most successful.