Jan 05, 2026
Daphnia magna: DNA extraction for downstream whole genome NGS
- Albano Pinto1,
- Joana Luísa Pereira1
- 1CESAM - Centre for Environmental and Marine Studies, Department of Biology, University of Aveiro, Portugal
Protocol Citation: Albano Pinto, Joana Luísa Pereira 2026. Daphnia magna: DNA extraction for downstream whole genome NGS. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv556j6v1b/v1
Manuscript citation:
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: Working
We use this protocol and it's working
Created: December 23, 2025
Last Modified: January 05, 2026
Protocol Integer ID: 235705
Keywords: kit-based DNA extraction, Daphnia, microcrustaceans, high quality DNA, dna from the freshwater microcrustacean daphnia magna, freshwater microcrustacean daphnia magna, dna extraction for downstream whole genome ng, dna extraction, extraction of dna, masterpure complete dna, level dna methylation assessment, downstream whole genome ng, genome sequencing, rna kit, sufficient dna yield, procedures for the extraction, sequencing application, dna, biosearch technology, extraction, rna, sequencing
Funders Acknowledgements:
European Union
Grant ID: 101078991
FCT
Grant ID: 2022.10817.BD
Abstract
This protocol provides optimised procedures for the extraction of DNA from the freshwater microcrustacean Daphnia magna, based on the use of a commercial kit (MasterPure Complete DNA and RNA Kit, Epicentre, Biosearch Technologies), added indications for preliminary quality control. By running this protocol, one should obtain sufficient DNA yield towards downstream next-generation whole-genome sequencing applications, e.g. base-level DNA methylation assessment (Methyl-seq); as well, samples should withstand good quality levels for the purpose, namely low-to-minimal contaminants and high molecular weight (low fragmentation).
Materials
Apart from the MasterPure Complete DNA and RNA Kit (Epicentre, Biosearch Technologies), the following materials will be needed to carry out the protocol:
Isopropanol, 99.5%, molecular biology gradeThermo Fisher ScientificCatalog #327272500
Ethanol 70%
SYBR Safe DNA Gel StainInvitrogen - Thermo Fisher
10X TBE BufferThermo Fisher Scientific
6X DNA Loading DyeThermo Fisher Scientific
GeneRuler 1 kb Plus DNA LadderThermo FisherCatalog #SM1331
Sterile 1.5-mL Eppendorf tubes
Sterile 15 mL Falcon tubes
Sterile pipette tips
Sterile pestles matching 1.5-mL Eppendorf tubes
Ice
Thermoblock set to 65°C
Refrigerated centrifuge (4ºC)
Microcentrifuge for short spins
Vortex
Qubit fluorometer (Qubit 3.0, Invitrogen)
Nanodrop spectrophotometer (NanoDrop 1000 spectrophotometer, Nano-Drop Technologies, Wilmington, DE, USA)
Electrophoresis system
Daphnids
Protocol materials
Isopropanol, 99.5%, molecular biology gradeThermo Fisher ScientificCatalog #327272500
Ethanol 70%
SYBR Safe DNA Gel StainInvitrogen - Thermo Fisher
6X DNA Loading DyeThermo Fisher Scientific
GeneRuler 1 kb Plus DNA LadderThermo FisherCatalog #SM1331
10X TBE BufferThermo Fisher Scientific
Troubleshooting
Before start
Note the materials, equipment and conditions required for the protocol, especially those items that are required apart from the kit. Ensure also the proper environment to carry out the protocol, including disinfected surfaces, established flows in handling and transfer of samples to prevent cross-contamination, availability of general labware, including disposal vessels, sterile vessels, tube holders as needed, labelling material, gloves, etc.
Tyssue lysis and digestion
5h 41m 20s
Prepare the lysis solution: dilute 1 μl of Proteinase K (50 μg/μl) into 300 μl of Tissue and Cell Lysis Solution (both supplied by the kit) per sample.
5m
Add 150 μl of Tissue and Cell Lysis solution with proteinase K to each tube with the Daphnids and grind the daphnids using a pestle.
20s
Add more 150 μl of Tissue and Cell Lysis solution with proteinase K to each Daphnids tube and mix by inverting the tubes several times.
30s
Incubate the Daphnids tubes at 65°C for 15 min in the thermoblock, and vortex the tubes every 5 minutes to mix.
20m
Cool the Daphnids down to 37°C
20m
Add 1 μl of 5 μg/μl RNAse A (supplied by the kit) to each sample, then mix by inverting the tubes several times. Spin-down the samples.
2m
Incubate the Daphnids tubes at 50°C for 3 hours in the thermoblock.
3h
Incubate the Daphnids tubes on ice for 5 minutes.
5m
Add 150 μl of MPC protein precipitation solution (supplied by the kit) to each Daphnids tube. Mix by vortexing for 10 seconds.
30s
Centrifuge at 4 °C for 10 minutes at 10000xg.
15m
Discard the pellet (do not disturb the pellet) and transfer the supernatant to new sterile 1.5-mL eppendorf tubes.
Add 500 μl of Isopropanol, 99.5%, molecular biology gradeThermo Fisher ScientificCatalog #327272500 . Mix by gently inverting 40 times.
3m
Centrifuge at 4 °C for 10 minutes at 13,000 rpm.
15m
Discard the supernatant (isopropanol) using a micropipette, carefully to not disturb the pellet. Centrifuge the tubes again for 10 seconds at room temperature and remove the remaining supernatant with a smaller bore pipette tip. Check if the DNA pellet (white) is visible.
Add 500 μl of refrigerated Ethanol 70% , gently, to avoid lifting the pellet. Spin-down the tubes at 4 °C if the pellet is loose. Discard the supernatant (ethanol) using a micropipette. Repeat this step 2 times.
5m
Dry the pelleted DNA at RT for 30 minutes in the tubes with the lids open.
30m
Resuspend the DNA with 35-37 μl of autoclaved UP water. Leave the DNA at 4 °C for 30 minutes to aid in DNA suspending.
35m
Aliquot the samples (≈15 µl) for quality control checks.
5m
Store DNA at -80°C or keep it at 4°C if preservation for a short period is intended before further steps.
gDNA quantity and quality check
1h 25m 20s
Run DNA quantification using fluorometry (recommended)
Prepare a 1-20 μL aliquot of sample (commonly 2 μL) and add the proprietary kit reagent for DNA quantification (dsDNA BR/HS) with Qubit to complete a final reading volume of 200 μL. For samples starting from higher biomass dilution might be needed before this step (check the expected DNA concentration vs. reading range).
5m
Read the solution in the Qubit for DNA quantification. The outcome will be the DNA concentration of the sample in ng/μL. If sample dilution applied, mind it to record the DNA concentration of the sample. For the reference samples as indicated in this protocol (Daphnids ), the expected DNA concentration is generally within 5-10 ng/μL.
20s
Run DNA quality screening using spectrophotometry
Set the Nanodrop spectrophotometer software for nucleic acids assessment
2m
Pipette a drop (2 μL recommended volume) of the sample into the holder in the reader. No dilution is needed if using similar biomass as indicated for the reference sample in this protocol (Daphnids )
30s
Read the sample. The outcome will be:
(i) Total nucleic acids concentration, typically within the range of 150-300 ng/μL if using samples similar to the reference sample in this protocol (Daphnids ). As there is no consistent quantification of gDNA concentration only, this concentration is not particularly informative.
(ii) Absorbance ratio 260/280 (~1.8 should be the target; appreciably lower ratios may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm). With this protocol, similar samples to that indicated as a reference (Daphnids ) and using the indicated kit, ratios around 1.9 are commonly reached.
(iii) Absorbance ratio 260/230 (2.0-2.2 should be the target; appreciably lower ratios indicate the presence of contaminants which absorb at 230 nm, such as EDTA, carbohydrates, phenol and some extraction reagents, e.g. TRIzol). With this protocol, similar samples to that indicated as a reference (Daphnids ) and using the indicated kit, ratios around 2.0 can be reached, but more commonly the ratios are within 1.7-1.9; this poses little to no issues in most downstream NGS applications.
30s
Clean and repeat the read at least once for consistency check. Some variability in readings is relatively common.
2m
Run DNA quality screening via electrophoresis
Prepare a 0.8% agarose gel stained with Invitrogen SYBR Safe DNA Gel StainInvitrogen - Thermo Fisher , following the manufacturer instructions.
20m
Place the gel in an electrophoresis tank filled with an appropriate electrophoresis buffer (e.g. 10X TBE BufferThermo Fisher Scientific , diluted to 1x) and load the samples with an appropriate loading dye (e.g., 6X DNA Loading DyeThermo Fisher Scientific , diluted to 1x). In the first slot add the ladder (e.g., GeneRuler 1 kb Plus DNA LadderThermo FisherCatalog #SM1331 ).
10m
Run an electrophoresis at 100V for 30 min.
40m
Take your gel to a gel documentation platform and check the bands pattern. Ideally, integer DNA fragments should be on the top of the gel, above the 10,000 bp ladder band.
5m
Acknowledgements
This protocol was optimised and elaborated under the scope of the project EPIBOOST, funded by the European Union (Grant 101078991; doi/org/10.3030/101078991). Views and opinions expressed are however those of the authors only and do not necessarily reflect those of the EU or the European Research Executive Agency; neither the EU or the granting authority can be held for them. Albano Pinto is grateful to FCT for the awarded individual PhD grant with the reference 2022.10817.BD and DOI identifier: https://doi. org/10.54499/2022.10817.BD.