Feb 25, 2026
Version 2
PCR amplification of enviromental DNA (eDNA) with barcoded primers for 16S/18S metabarcoding V.2
This protocol is a draft, published without a DOI.
- June Sales1,
- Hannah Benisty1
- 1Centre for Genomic Regulation
- PyriSentinel
Protocol Citation: June Sales, Hannah Benisty 2026. PCR amplification of enviromental DNA (eDNA) with barcoded primers for 16S/18S metabarcoding. 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: February 25, 2026
Last Modified: February 26, 2026
Protocol Integer ID: 244004
Keywords: pcr amplification of enviromental dna, biodiversity from environmental dna, environmental dna, dna extraction, lake water sample, barcoded primer, biodiversity, pcr amplification, rrna gene, pcr, metabarcoding protocol, pyrenee, primer, dna extraction from those lake water sample, pcr amplification of enviromental dna, biodiversity from environmental dna, environmental dna, dna extraction, lake water sample, enviromental dna, barcoded primer, biodiversity, pcr amplification, rrna gene, 16s rrna gene, metabarcoding protocol, pyrenee
Funders Acknowledgements:
Interreg VI-A Spain-France-Andorra Program (POCTEFA 2021-2027)
Grant ID: PyriSentinel - EFA059/01
Abstract
We present a metabarcoding protocol to assess biodiversity from environmental DNA (eDNA) previously extracted from ~300 lake water samples from the Pyrenees. Two independent libraries are prepared: one targeting the 16S rRNA gene (prokaryotes) and one targeting the 18S rRNA gene (eukaryotes). Samples were labeled with barcoded primers to distinguish them during sequencing. PCR amplification incorporates both the target sequence and barcode, allowing all samples to be pooled into two final libraries for sequencing.
In the Pyrisentinel workspace, two additional protocols are available that describe in detail the procedures for sampling and DNA extraction from those lake water samples collected in the Pyrenees.
Attachments
Materials
- Barcoded primers (synthesized by IDT)
- Sample material
- Ultrapure water
- KAPA HiFi Hot Start Ready Mix
- Agarose
- TAE buffer
- SYBR Safe DNA Gel Stain
- AMPure XP Beads
- Freshly prepared 70% Ethanol
- PCR tubes
- Capped strips
- 1.5 mL eppendorf tubes
- Electrophoresis chamber
- Transilluminator
- Magnetic rack
- Pipettes
- Pipette tips
- UV hood
- Thermocycler
- Vortex
- Thermomixer
- Benchtop centrifuge
Before start
All steps should be performed inside a clean hood to minimize the risk of contamination.
Barcode design
Prepare primers with barcodes as detailed in the attached file before starting the PCRs.
Reagents and sample preparation, workspace setup
30m
Irradiate the clean hood with UV light for 00:30:00 .
30m
Thaw all reagents and samples on ice, preparing enough volume for the number of PCRs to be performed.
PCR Mix Preparation
1h
Dilute extracted DNA samples with ultapure water to achieve a final concentration of 5 - 10 ng/µL . Keep the sample dilutions on ice.
10m
Prepare the PCR mastermix, adding enough volume for the desired number of samples. Include extra volume equivalent to one reaction to minimize pipetting errors. The volumes for a single PCR reaction are listed in the table below. Keep the prepared mastermix on ice until use.
| Reagent | Volume (µL) | |
| Water | 5.5 | |
| KAPA | 12.5 |
10m
Each PCR sample will be performed individually in strip tubes. Label the strip tubes and add 18 µL of the prepared mastermix to each.
15m
Add 6 µL of the appropriate combination of primers/barcodes (3 µL of the forward primer and 3 µL of the reverse primer) to the corresponding strip tube for each PCR sample.
10m
Add 1 µL of the diluted DNA sample to the corresponding strip tube.
10m
Mix the reaction by pipetting and split the total volume into three separate tubes. Spin down.
5m
PCR
1h 30m
Program the thermocycler according to the following cycling conditions.
| Step | Temperature (°C) | Time (s) | Cycles | |
| Initial denaturation | 95 | 180 | 1 | |
| Denaturation | 98 | 20 | 22 | |
| Annealing | 57 | 30 | ||
| Extension | 72 | 75 | ||
| Final extension | 72 | 300 | 1 | |
| Hold | 4 | - | - |
1h 30m
Gel electrophoresis
55m
Once the PCR is complete, pool the three previously split PCR products into a single tube per sample.
5m
Load 4 µL of each sample with 1 µL of loading buffer onto a 1% agarose gel containing a preferred DNA stain, and run electrophoresis at 100 V for 00:30:00 .
40m
Visualize the gel results using a transilluminator and discard PCR products from samples that did not amplify.
Note
For samples that did not amplify, optimization may be achieved either by increasing the DNA input (e.g., adding 2 µL of DNA) or by further diluting the template. These adjustments should be tested on problematic samples.
10m
PCR purification
2h 5m
Take an aliquot of beads out of the fridge and leave at room temperature for 00:30:00 .
30m
Add 19 µL of ultrapure water to each PCR product that amplified to reach a final volume of 40 µL , as the PCR product volume after gel is 21 µL .
5m
Vortex the beads aliquot briefly and add 32 µL of beads (0.8X volume) to the PCR sample. Spin down.
10m
Place the tubes on a shaker at 350 rpm for 00:10:00 .
10m
Spin down and place the tubes on a magnetic rack for 00:05:00 until the beads are completely collected on the side of the tube.
8m
Carefully remove and discard the supernatant without disturbing the beads.
5m
Add 200 µL of freshly prepared 70% ethanol to de beads without disturbing them. Incubate 00:00:30 and remove the ethanol with a pipette.
5m
Repeat the previous step.
5m
Spin down the beads and remove any remaining ethanol with a pipette. Allow the beads air-dry at room temperature for 00:03:00 .
Note
Do not overdry the beads; stop drying before cracks appear.
7m
Elute the DNA by adding 14 µL of ultrapure water to the beads. Place the tubes on a shaker at 350 rpm and 37 °C for 00:05:00 .
10m
Place the tubes on a magnetic rack for 00:03:00 . Transfer the supernatant to a new tube. Discard the beads.
10m
Mesure the concentration of the obtained DNA using a Qubit fluorometer.
20m
Protocol references
Latz, M. A. C., Grujcic, V., Brugel, S., Lycken, J., John, U., Karlson, B., Andersson, A., & Andersson, A. F.(2022). Short- and long-read metabarcoding of the eukaryotic rRNA operon: Evaluation of primers and comparison to shotgun metagenomics sequencing. Molecular Ecology Resources, 22, 2304–2318. https://doi.org/10.1111/1755-0998.13623
Lara Urban, Andre Holzer, J Jotautas Baronas, Michael B Hall, Philipp Braeuninger-Weimer, Michael J Scherm,
Daniel J Kunz, Surangi N Perera, Daniel E Martin-Herranz, Edward T Tipper,
Susannah J Salter, Maximilian R Stammnitz (2021) Freshwater monitoring by
nanopore sequencing eLife 10:e61504 https://doi.org/10.7554/eLife.61504
Balzano S, Abs E, Leterme SC (2015) Protist diversity along a salinity gradient in a
coastal lagoon. Aquat Microb Ecol 74:263-277 https://doi.org/10.3354/ame01740
PacBio 16S Kinnex: Preaparing Kinnex libraries from 16S rRNA amplicons: https://www.pacb.com/wp-content/uploads/Procedure-checklist-Preparing-Kinnex-libraries-from-16S-rRNA-amplicons.pdf