DNA Extraction from Archival Formalin-fixed, Paraffin-embedded Tissue Sections

DNA Extraction from Archival Formalin-fixed, Paraffin-embedded Tissue Sections

Section 1. Non-heating DNA Extraction Protocol

1. Cut paraffin block at 10 µm and collected in an autoclaved plastic microtube (1.5 ml).
2. Add 1 ml xylene to the microtube containing tissue sections for 30 min for two changes,
3. Add 100% and 75% ethanol for 30 min with two changes
4. Wash with PBS for 15 min with two changes
5. Add 500 µl of lysis buffer (proteinase K 20 mg/ml, 50 µl, 1 M Tris-HCl solution 10 µl, 0.5 M EDTA 2 µl, 10% SDS 100 µl, and distilled water 838 ml)
6. Incubated at 52° C overnight until all tissue fragments were dissolved completely
7. Add 500 µl phenol:chloroform:isopropanol alcohol at 25:24:1 to the de-waxed tissue
8. Mix by vortex
9. Centrifugation at RT, 12,000 x g for 10 min
10. Transfer the supernatant fluid to an autoclaved microtube using a 100-µl pipette
11. Add one volume of chloroform to the supernatant, mixed by vortexing
12. Centrifuged at 12,000 x g for 5 min.
13. Carefully remove the upper aqueous supernatant to another fresh microtube
14. Adding 0.1 volume of 3 M sodium acetate to the new tube
15. Mix by vortexing
16. Add 1 volume of isopropanol, and incubate at -20C overnight.
17. The precipitated DNA was centrifuged at 12,000 x g at 4C.
18. Discard the supernatant fluid and wash once with 75% ethanol.
19. Collect the extracted DNA after further centrifugation.
20. Dissolve the final yield of DNA in 50 µl distilled water after drying completely in a hood.