Nov 11, 2025

eDNA Extraction from Pyrenees High-Mountain Lake Samples Using the Sylphium Kit V.1

This  protocol  is a draft, published without a DOI.
eDNA Extraction from Pyrenees High-Mountain Lake Samples Using the Sylphium Kit
  • June Sales1,
  • hannah.benisty 1
  • 1Centre for Genomic Regulation
  • PyriSentinel
Hannah Benisty
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External link: https://pyrisentinel.eu
Protocol Citation: June Sales, hannah.benisty 2025. eDNA Extraction from Pyrenees High-Mountain Lake Samples Using the Sylphium Kit. protocols.io https://dx.doi.org/Version created by June Sales
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: November 10, 2025
Last Modified: February 24, 2026
Protocol  Integer ID: 231987
Keywords: dna extraction, extraction of dna, sylphium extraction kit, lake water sample, extraction from pyrenees high, mountain lake sample, dna fragment, water sample, using sylphium brand filter, quality dna, dna, sequencing technology, quality dna suitable for subsequent genomic analysis, sylphium brand filters with pore size, extraction, sylphium kit, sylphium kit this protocol, dna with the greatest possible length
Funders Acknowledgements:
Interreg VI-A Spain-France-Andorra Program (POCTEFA 2021-2027)
Grant ID: PyriSentinel - EFA059/01
Abstract
This protocol describes the extraction of DNA from lake water samples collected in the Pyrenees as part of the PyriSentinel project. Water samples were filtered using Sylphium brand filters with pore sizes of 500 µm and 22 µm. DNA extraction was performed using the Sylphium extraction kit, chosen to preserve DNA with the greatest possible length. Preserving fragment length is important for downstream applications involving long-read sequencing technologies such as Nanopore and PacBio. This method yields high-quality DNA suitable for subsequent genomic analyses.
Materials
70% ethanol
Sylphium Enviromental DNA Isolation Kit (https://sylphium.com/webshop/product/syl002/)
  • S2 (Chloroform)
  • S3 (Precipitation Buffer)
  • S4 (Isopropanol)
  • S5 (80% ethanol)
  • S6 (Conservation buffer)
Qubit dsDNA Quantification Assay Kit (https://www.thermofisher.com/order/catalog/product/es/en/Q32851)
Syringes
2 mL microcentrifuge tubes
1.5 mL microcentrifuge tubes
15 mL falcon
Pipette tips (p10, p20, p200, p1000)
Qubit tubes


Before start
All steps should be performed inside a clean hood to minimize the risk of contamination. When handling chloroform, work must be carried out within the fume hood.
Protocol overview


Sample Preparation and Work Area Setup
1h
Remove filters stored at -70 °C and incubate at 60 °C for 00:30:00 to thaw the sample material.
30m
Disinfect the hood surface by wiping with 70% ethanol, followed by UV irradiation for 00:15:00 .
15m
Prepare the necessary materials for the extraction (eppendorf tubes, falcon tubes and syringes) inside the hood, label each item accordingly, and irradiate with UV light for an additional 00:15:00 .
15m
eDNA extraction
Briefly vortex the thawed filter and clean its exterior surface with 70% ethanol before handling it inside the hood.
Unscrew the cap of the filter indicated by the arrow. Attach the syringe to that end and extract the content from inside the filter. Transfer the extracted liquid into a 15 mL Falcon tube. Repeat the extraction with the syringe up to three times to ensure complete recovery of the filtrate.




3m
Record the total volume extracted from the filter.

Expected result
Between approximately 3 and 4 mL. Volumes around 6 mL may indicate dilution of the preservation buffer and could result in very low DNA concentrations.

1m
Inside the fume hood, add 750 µL of Solution S2 (Chloroform) to the Falcon tube containing the filtrate. Gently shake the two phases, avoiding vigorous movements that could shear the DNA.

Safety information
Chloroform is a hazardous chemical and must be handled with care. It is toxic by inhalation, ingestion, and skin absorption, and is also a suspected carcinogen. Always use chloroform inside a certified fume hood while wearing appropriate personal protective equipment (PPE), including a lab coat, safety goggles, and chemical-resistant gloves. Avoid direct contact and prolonged exposure. Dispose of waste according to institutional and local hazardous waste disposal guidelines.

5m
Centrifuge the falcon tube containing the filtrate and Solution S2 (chloroform) for 00:15:00 at 3180 rpm, 17°C .

Note
After centrifugation, three distinct phases should be visible. The upper aqueus phase is the polar phase, which contains the DNA to be isolated.



















15m
While centrifuging the falcon tubes, prepare the precipitation solution inside a clean hood. For each filter, take two 2 mL microcentrifuge tubes that have been previously UV-irradiated. Add 100 µL of Solution S3 (precipitation buffer) and 900 µL of Solution S4 (isopropanol) to each tube. Once prepared, set the tubes aside until needed in the next step.
Inside the fume hood, proceed with the centrifuged tubes, in which three distinct phases should now be visible. Carefully collect 1000 µL of the upper (aqueous) phase and transfer it into one of the precipitation solution tubes prepared in the previous step. Repeat the same for the second precipitation tube. Shake the tubes gently.
5m
Incubate the precipitation mixtures at -20 °C for 01:00:00 .
1h
Set the microcentrifuge to 4 °C .
20m
Centrifuge the precipitation tubes at 16100 rpm and 4 °C for 00:45:00 .
45m
After centrifugation, a white pellet will form at the bottom of each tube. Carefully discard the supernatant from both tubes without disturbing the pellets.
5m
Add 500 µL of Solution S5 (80% ethanol) to each of the two tubes. Centrifuge at 16100 rpm and 4 °C for 00:05:00 . Discard supernatant.

Note
It is important to keep Solution S5 at -20 °C to prevent the pellet from dissolving.


7m
Repeat the previous step.
7m
At this point, each of the two tubes should contain a pellet. Let the pellets air-dry at room temperature inside the clean hood for 15 minutes.
Note
The time of drying of the pellet can vary depending on the temperature of the laboratory, we will know that it is dry when it turns transparent.

15m
Add 37 µL of Solution S6 (conservation buffer) to each of the two tubes containing the dried pellet.

Note
IMPORTANT: The preservation buffer contains EDTA. If the presence of EDTA is a concern for downstream applications, you may replace the preservation buffer with 10 mM Tris-HCl buffer, pH 7.5–8.

3m
To dissolve the pellet in the storage buffer, place the tubes in a thermomixer set to Room temperature and shake at 350 rpm for 00:10:00 .

10m
After the incubation time, combine the contents of the two tubes into a single 1.5 mL microcentrifuge tube. The final volume of eluted DNA should be 74 µL.


Note
The final volume after the quantification will be 70 µL.

3m
Extracted DNA quantification
16m
DNA quantification using Qubit. Prepare and label the tubes required (200 µL tubes).
3m
Prepare the working solution in a 1.5 mL tube by diluting the Qubit reagent 1:200 in the Qubit buffer (for example for 1 sample would be 1 µL of reagent and 199 µL of buffer). Mix using vortex.

3m
For each quantification reaction, add 199 µL of the Qubit working solution to the quantification tubes, followed by 1 µL of the DNA sample. Vigorously vortex. Proceed to read the samples.

5m
Also quantify the DNA sample using NanoDrop. Use 1-2 µL of undiluted DNA sample directly on the measurement pedestal. Record the DNA concentration (ng/µL) as well as the quality ratios A260/A280 and A260/A230. Clean the pedestal.
5m
Protocol references