Sakata et al. Fish SedDNA Extraction Protocol
Masayuki K. Sakata
Abstract
Variations of this standardised protocol have been used by Masayuki K. Sakata and colleagues to successfully extract fish eDNA from modern and historic Japanese lake and river sediments.
The method has been used to recover fish species composition data from modern surface sediments from a lake (Sakata et al., 2020) and a small, natural river (Sakata et al., 2021). It has also been applied to detect target fish species in lake sediments up to 100 years old (Sakata et al., 2022).
This extraction method represents a consolidation of the methods applied in the following publications:
- Sakata, M. K., Yamamoto, S., Gotoh, R. O., Miya, M., Yamanaka, H., & Minamoto, T. (2020). Sedimentary eDNA provides different information on timescale and fish species composition compared with aqueous eDNA. Environmental DNA, 2(4),
505– 518. https://doi.org/10.1002/edn3.75
- Sakata, M. K., Watanabe, T., Maki, N., Ikeda, K., Kosuge, T., Okada, H., … Minamoto, T. (2021). Determining an effective sampling method for eDNA metabarcoding: a case study for fish biodiversity monitoring in a small, natural river.
Limnology, 22(2), 221–235. https://doi.org/10.1007/s10201-020-00645-9
- Sakata, M.K., Tsugeki, N., Kuwae, M., Ochi, N., Hayami, K., Osawa, R., Morimoto, T., Yasashimoto, T., Takeshita, D., Doi, H., & Minamoto, T. (2022). Fish environmental DNA in lake sediment overcomes the gap of reconstructing past fauna
in lake ecosystems. bioRxiv, https://doi.org/10.1101/2022.06.16.496507
Steps
Alkaline extraction
PLACE 9.0g of sediment sample into a 50 mL tube
ADD 6mL NaOH
ADD 3mL TE buffer
ADD 500µL G2 enhancer
VORTEX samples to mix
INCUBATE samples at 94°C for 0h 50m 0s
ALLOW samples to cool to Room temperature
CENTRIFUGE at 5000x g for 0h 0m 30s
TRANSFER 7.5mL of supernatant to a new 50 mL tube
NEUTRALIZE by adding 7.5mL Tris-HCL (1M)
Ethanol precipitation
ADD 1.5mL sodium acetate
VORTEX well to mix
ADD 30mL of ethanol
VORTEX well to mix
INCUBATE at -20°C for at least 1h 0m 0s
WIPE off condensation from around the 50 mL tube and centrifuge at 5,350xG for 20 min
CENTRIFUGE at 5350x g for 0h 20m 0s
DISCARD supernatant and leave the tube mouth down for a few minutes
TRANSFER the precipitate to a PowerBead Tube (Qiagen Kit)
DISSOLVE the remaining precipitate with 100µL of DW then transfer to the PowerBead Tube
DNeasy PowerSoil extraction
ADD 60µL of Solution C1
VORTEX at max speed for 0h 10m 0s
CENTRIFUGE at 10000x g for 0h 0m 30s
TRANSFER supernatant to a new 2 mL tube
ADD 250µL of Solution C2
VORTEX briefly to mix
INCUBATE at 4°C for 0h 5m 0s
CENTRIFUGE at 10000x g 0h 1m 0s
TRANSFER 600µL of supernatant to a new 2 mL tube
ADD 200µL of Solution C3
VORTEX briefly to mix
INCUBATE at 4°C for 0h 5m 0s
CENTRIFUGE at 10000x g for 0h 1m 0s
TRANSFER 750µL of supernatant to a new 2 mL tube
ADD 1.2mL of Solution C4
VORTEX well to mix
TRANSFER 675µL to a Spin Filter
CENTRIFUGE at 10000x g for 0h 1m 0s
DISCARD the liquid filtrate
REPEAT the above step until all liquid has passed through the Spin Filter
ADD 500µL of Solution C5 to the Spin Filter
CENTRIFUGE at 10000x g for 0h 0m 30s
DISCARD the liquid filtrate
TRANSFER the Spin Filter to a new 1.5 mL tube
ADD 100µL of Solution C6 to the Spin Filter
LET stand for 0h 1m 0s
CENTRIFUGE at 10000x g for 0h 0m 30s
DISCARD the Spin Filter
DNA is now ready for downstream applications
