TITLE: DNA Extraction for Formalin Specimen
AUTHORS: heeseung

[DESCRIPTION]
DNA Extraction for Formalin Specimen (Qiagen Kit: QIAamp DNA FFPE Advanced Kit)

[STEPS]
SECTION: Deparaffinization process
1. 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). 
SECTION: Deparaffinization process
2. Incubate at 56°C for 3 min, then allow to cool to room temperature.
SECTION: Preparation for Extraction 1
3. 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.
SECTION: Preparation for Extraction 2
6. 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. 
SECTION: Preparation for Extraction 2
7. Add 2 μl RNase A, vortex, and incubate for 2 min at room temperature on the bench.
SECTION: Preparation for Extraction 2
8. Add 20 µl Proteinase K, vortex, and incubate for 15 min at 65°C and 450 rpm.
SECTION: Preparation for Extraction 2
9. Add 250 μl Buffer AL and 250 μl ethanol (96–100%) to each sample and mix thoroughly by vortexing
SECTION: Extraction
10. 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.
SECTION: Extraction
11. 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.
SECTION: Extraction
12. 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.
SECTION: Extraction
13. 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.
SECTION: Extraction
14. 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.
SECTION: Extraction
15. 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.
SECTION: Extraction
16. 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.
SECTION: Lysis
4. 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".
SECTION: Lysis
5. Incubate for 1 h at 90°C without shaking.