Jun 25, 2026
Chelex-100 eDNA Extraction Protocol (beta)
This protocol is a draft, published without a DOI.
- 1Independent
Protocol Citation: Matthew Penney 2026. Chelex-100 eDNA Extraction Protocol (beta). protocols.io https://dx.doi.org/
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
The protocol results in a usable sample, but it is not optimized and long-term sample stability is unknown.
Created: June 08, 2026
Last Modified: June 25, 2026
Protocol Integer ID: 318660
Keywords: eDNA, Chelex-100, Beta, Low-Cost, Salmo salar, environmental DNA, Artificial Samples, Atlantic Canada, edna filter, water sample, extraction protocol, artificial sample, results in usable sample, sample, atlantic salmon, filtering 1l amount, usable sample, grade water
Disclaimer
This protocol was tested in the Marine Gene Probe Lab at Dalhousie University (PI: Dr. Paul Bentzen) using their reagents and laboratory spaces. Bioinformatic work was performed on these samples by the Research Associate (Kristin Watson), though results are not discussed here.
Abstract
A Chelex-100 extraction protocol for eDNA filters from water samples. This was designed for and tested on artificial samples made by diluting water from a tank of Atlantic Salmon (Salmo salar) into molecular-grade water and filtering 1L amounts. Results in usable samples and is cheaper than using a Qiagen kit, but long-term sample quality has not been assessed. Currently only recommended for samples you do not intend to re-visit.
Materials
For this protocol, you will need:
Reagents/Solutions
-10% Chelex-100 Solution
-Proteinase K (600-1000U/ml)
-7.5M Ammonium acetate
-3M Sodium acetate
-70% Ethanol solution, room temperature (for washing)
-100% Ethanol solution, ice-cold (for precipitation)
-80100% Ethanol solution, room temp (for flame sterilization)
Plastics/Equipment
-2ml lock-cap microcentrifuge tubes
-1.5ml sterile microcentrifuge tubes*
-Dry or hot water bath, or incubator
-Metal forceps (NOT plastic)
-Bunsen burner or utility lighter
*For improved long-term sample quality, you can use a Low-Bind 1.5ml microcentrifuge Tube (Sarstedt) for the elution step. They do cost more, but you will lose less of your sample to stochastic binding over time.
Troubleshooting
Problem
Trouble Seeing The Pellet
Solution
When centrifuging, place the tubes in the centrifuge wells with the hinges facing outward from the center and perpendicular to the edge of the rotator. This will result in the pellet, even if not visible, being located in a predictable spot.
Safety warnings
Do not perform this protocol in labs where tissue or genomic DNA samples are currently being stored or processed. If they have been previously, thoroughly decontaminate the lab space with a sterilizing agent (e.g. DNA Away, 0.3% bleach) and run a blank test before processing any eDNA samples.
And, as always, no food or drink in the lab. Along with being bad practice, bringing in food or drink might contaminate your samples (yes, really).
Ethics statement
The salmon water sourced in this study were held in the Dalhousie University Aquatron, overseen by a dedicated Animal Care team. No salmon were harmed during the sourcing of the water.
Before start
Prior to the extraction, it is VERY important to ensure that you are working in a laboratory space which is specifically dedicated to eDNA work (i.e. no genomic DNA or tissue samples enter the lab at any point, nor do any individuals who have potentially been contaminated) and has been properly decontaminated prior to the extraction.
You must also ensure that you have a sterile, dry compartment in which to dry off samples prior to elution. It must be free from outside airflow (source of contamination) as the tubes must remain open during the drying stage. A sterilized drawer is an option for this, ideally one not under or near your lab bench (bleach fumes may damage the DNA during the drying step).
To clean surfaces, dilute a bleach solution to 0.3%. If you do not know the exact concentration of your bleach, dilute 1/10.
Using a sponge or cloth, wipe down all lab surfaces with the bleach solution and let stand for at least 5 minutes. Following this, go over the surfaces again with a damp cloth (just water) to wipe off residual bleach. From here, allow surfaces to air-dry to completion for at least one hour.
*To avoid losing time to this, I recommend bleaching your lab after every safe stop point. It gives the lab an entire night to dry.
Initial Notes
Based on the following articles:
- Singh, U.A., Kumari, M., and S. Iyengar. 2018. Method for improving the quality of genomic DNA obtained from minute quantities of tissue and blood samples using Chelex 100 resin. Biological Procedures Online. 20(12)
- Bockrath, K.D., Tuttle-Lau, M., Mize, E.L., Ruden, K.V., and Z. Woiak. 2022. Direct comparison of eDNA capture and extraction methods through measuring recovery of synthetic DNA cloned into living cells. Environmental DNA. 4(5)
And with considerations from:
- Simon, N., Shallat, J., Wietzikoski, C.W., and W.E. Harrington. 2020. Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots. Biology Methods and Protocols. 5(1)
For use to extract environmental DNA from filters, mainly for water samples. This protocol has been tested once using artificial samples made by diluting water from a tank of Atlantic Salmon (Salmo salar) in molecular-grade water and filtering 1L samples in triplicate. This extraction protocol resulted in samples which were successfully assayed with qPCR and sequenced. It is significantly cheaper per sample than using a Qiagen kit, but is not scalable for high-throughput operations, has more overnight steps, and long-term sample quality has not been assessed. Only recommended in its current state for samples you plan to assay/sequence once and not re-visit.
Buffer Preparations
Chelex-100 Buffer (10% solution)
Components:
- Chelex-100 (Bio-Rad recommended)
- 1X TE
Dilute one part Chelex-100 in 9 parts 1X TE. Mix well.
7.5M Ammonium Acetate (500ml)
Components:
- Ammonium acetate, powdered (97% pure at least)
- Molecular-Grade H2O
Steps:
- Add a small amount of the water to a beaker with a maximum volume of 1L (to avoid spillage). If you have a magnetic stirring stand, insert a stir bar.
- Add 289.05g of ammonium acetate powder to the beaker.
- Add molecular-grade water until your meniscus hits the 500ml line. Mix in the powder until fully dissolved.
To make different amounts:
-For each liter of water, you would add 578.1g of Ammonium Acetate powder. Adjust the grams you add to the volume of water you wish to use.
3M Sodium Acetate (500ml)
Components:
- Sodium Acetate powder, anhydrous
- Molecular-Grade H2O
Steps:
- Add a small amount of the water to a beaker with a maximum volume of 1L (to avoid spillage). If you have a magnetic stirring stand, insert a stir bar.
- Add 123.05g of ammonium acetate powder to the beaker.
- Add molecular-grade water until your meniscus hits the 500ml line. Mix in the powder until fully dissolved.
- If you need it at a certain pH, add a sterilized pH probe and adjust the pH using drops of acetic acid to lower the pH or sodium hydroxide to raise it.
*Admittedly, I'm still not sure what the best concentrations to use for adjustments are, but don't use undiluted solutions of either. The pH will change too fast.
To make different amounts:
-For each liter of water, you would add 246.09g of Sodium Acetate powder. Adjust the grams you add to the volume of water you wish to use.
NOTE: If you're not comfortable making these buffers yourself, you can buy them pre-made. Or offer your labmate one beer per solution they prep for you (not official advice).
eDNA Extraction Protocol
DNA Extraction (Day 1)
In a 2ml lock-cap microcentrifuge tube, fold and place your filter paper into the bottom of the tube using the tweezers (flame-sterilize them before handing the filter). Add 600ul Chelex (10% soln) and 3ul Proteinase K to the tube, snap closed, and then vortex or shake to mix.
Heat to 55oC and incubate overnight.
*Safe stop point
DNA Extraction (Day 2)
*Before starting this step, make sure to sterilize your lab bench at least one hour before starting. Ideally, this is done the night before while samples are incubating. If using a bleach solution, do not initiate lab work until bleach smell is completely gone.
Increase the heat to 95oC and leave the reactions at this temperature for 4-5 minutes. This denatures the Proteinase K for the next step.
Spin the reactions down at 9000rpm for 1 minute. Transfer 200μL of supernatant to a new 1.5ml microcentrifuge tube.
To the new tube, add 100μL 7.5M ammonium acetate and vortex briefly to mix. Rest the reactions on ice for 5 minutes. Re-vortex briefly and then centrifuge 12000rpm for 10mins to pellet out any precipitated proteins. Avoiding the pellet (if present), transfer 250μL of supernatant to a new 1.5ml microcentrifuge tube. NOTE: this is the tube which will hold the finished sample.
Add 25μL 3M sodium acetate, pH 7.0, to the supernatant and briefly vortex to mix. Then add 550μL ice-cold 100% ethanol and vortex briefly again. Leave solution in a freezer (at least -20oC) overnight to precipitate the DNA.
*Safe stop point
DNA Extraction (Day 3)
*Before starting this step, make sure to sterilize your lab bench at least one hour before starting. Ideally, this is done the night before while samples are incubating. If using a bleach solution, do not initiate lab work until bleach smell is completely gone.
Remove the tubes from the freezer and centrifuge at 14,800rpm for 30 minutes to pellet out DNA.
*The pellet may be invisible to the naked eye, so insert tubes into the centrifuge with the hinges pointing away from the center of the centrifuge. This helps to predict where the pellet will be.
Decant tubes by pouring off solution and gently dabbing off excess liquid on a clean paper towel. Try to use the side which was not facing outward and don't touch the same spot twice to avoid cross-contamination.
After decanting, add 900μL of room-temperature 70% EtOH. Centrifuge at 14,800rpm for 5mins. Decant again, dab dry on a NEW clean cloth (don't use the same one), and dry the samples overnight in a sterile, isolated environment.
*Stop point, BUT do not bleach
DNA Extraction (Day 4)
*Before starting this step, sterilize your lab bench IF the drawer with your drying samples is not near or under it. As the tubes are currently open, the DNA is not protected.
If sample has dried completely, elute in 50ul of 1X TE buffer. From here, you may either agitate by vortexing gently to elute or leave the samples in the fridge. Overnight is ideal to ensure completion.
Thoughts & Considerations
-Based on my estimations, this protocol using Chelex-100 should cost (per sample) about 1/5 what an extraction with a Qiagen DNeasy kit costs.
-There seems to be some disagreement between Bockrath et al. (2022) and Simon et al. (2020) about
whether to do a high-temp extraction that goes quickly or a low-temp extraction that goes overnight. The low-temp variant uses Proteinase-K while the high-temp one does not (uses detergent instead). This is worth revisiting later since multiple overnight steps make a protocol undesirable.
-The protocol isn't timed since the time per step will ultimately be determined by how many samples you are processing together.
-The concentration of the Chelex-100 could also be revisited. Bockrath et al. is where the 10% was derived from, but Singh et al. goes as low as 5%. If the reaction works just as well at 5% there's no reason to keep using 10%.
Protocol references
- Singh, U.A., Kumari, M., and S. Iyengar. 2018. Method for improving the quality of genomic DNA obtained from minute quantities of tissue and blood samples using Chelex 100 resin. Biological Procedures Online. 20(12). doi: 10.1186/s12575-018-0077-6.
- Bockrath, K.D., Tuttle-Lau, M., Mize, E.L., Ruden, K.V., and Z. Woiak. 2022. Direct comparison of eDNA capture and extraction methods through measuring recovery of synthetic DNA cloned into living cells. Environmental DNA. 4(5): 1000-1010. https://doi.org/10.1002/edn3.330
And with considerations from:
- Simon, N., Shallat, J., Wietzikoski, C.W., and W.E. Harrington. 2020. Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots. Biology Methods and Protocols. 5(1): bpaa009. doi: 10.1093/biomethods/bpaa009.
Acknowledgements
Samantha Beal
Dr. Paul Bentzen
Kristin Watson
John Batt