DNA Extraction from Formalin-Fixed Tissues

As described in Gould et al. 2021; modified from Hykin et al. 2015 and Ruane & Austin, 2017

GHS Toxic While this protocol does not include any formalin or formaldehyde, seek appropriate training from your department or the CCG Lab Manager before working with or handling formalin-fixed tissues. Formalin/formaldehyde is a carcinogen, mutagen and toxic chemical that requires extra hazardous materials training per Cal Academy policy.

Gather the following materials:

  • GTE Buffer
  • 100% ethanol (EtOH), molecular-grade
  • 70% ethanol (EtOH), molecular-grade
  • Nuclease-free water
  • Qiagen DNeasy Blood & Tissue Kit

Protocol

  1. Aseptically dissect tissue and place into 1 ml of GTE buffer

  2. Soak for 3 hrs at room temperature

  3. Place tissue into 1 mL fresh GTE buffer and soak again for 3 hrs. at room temp.

  4. Transfer tissue into 1 mL of fresh GTE buffer and soak overnight (room temp.)

  5. The next morning preheat 180 ul aliquots of ATL buffer (QIAGEN) to 98°C

  6. Transfer tissue into 1 ml of 100% ethanol for 1 min at room temp.

  7. Transfer into 1 ml of 70% ethanol for 5 min at room temp.

  8. Transfer into 1 ml of nuclease-free water for 10 min at room temp.

  9. Transfer tissue into 180 ul of pre-heated (98°C) ATL buffer (QIAGEN)

  10. Incubate samples at 98°C for 15 min

  11. Immediately place samples on ice for at least 2 min.

  12. Once cooled, add 40 ul of proteinase K to each sample

  13. Vortex and incubate samples at 60°C for 48 hrs on a shaking heat block

  14. Vortex samples periodically and add additional 20 ul aliquots of proteinase K as needed to digest tissue (up to 100 ul total)

  15. Following this incubation period, extract DNA using the QIAGEN DNEasy Blood and Tissue Kit as described

  16. Elute DNA into 50 ul of nuclease-free water after a 3-min incubation at 55°C

References

Gould AL, Fritts-Penniman A, Gaisiner A. Museum Genomics Illuminate the High Specificity of a Bioluminescent Symbiosis for a Genus of Reef Fish. Front Ecol Evol. 2021 Feb;9:630207. doi: 10.3389/fevo.2021.630207

Hykin, S. M., Bi, K., and McGuire, J. A. (2015). Fixing formalin: a method to recover genomic-scale DNA sequence data from formalin-fixed museum specimens using high-throughput sequencing. PLoS ONE 10:e0141579. doi: 10.1371/journal.pone.0141579

Ruane, S., and Austin, C. C. (2017). Phylogenomics using formalin-fixed and 100+ year-old intractable natural history specimens. Mol. Ecol. Resources, 17, 1003–1008. doi: 10.1111/1755-0998.12655