May 04, 2026
Green microalgae: DNA extraction and preliminary quality control
- Inês Macário1,
- Joana Luísa Pereira1
- 1CESAM - Centre for Environmental and Marine Studies, Department of Biology, University of Aveiro, Portugal
Protocol Citation: Inês Macário, Joana Luísa Pereira 2026. Green microalgae: DNA extraction and preliminary quality control. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyoqkmgx9/v1
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
Created: March 15, 2026
Last Modified: May 04, 2026
Protocol Integer ID: 313295
Keywords: DNA extraction, green microalgae, Parachlorella, Chlorella, freshwater microalgae, high quality DNA, dna from green microalgae, green microalgae, chlorella vulgari, level dna methylation assessment, dna extraction, sufficient dna yield, sequencing application, dna, procedures for the extraction, methyl
Funders Acknowledgements:
European Union
Grant ID: 101078991
Abstract
This protocol provides optimised procedures for the extraction of DNA from green microalgae. We optimised it based on laboratory non-axenic cultures of cf. Chlorella vulgaris were used for the optimisation, grown in Woods Hole MBL medium at 20 + 2ºC under a photoperiod of 16hL:8hD provided by white cold light bulbs. Different culturing conditions and different species may require adjustments. 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 with low fragmentation.
Image Attribution
Funded by the European Union
Guidelines
The protocol refers to the materials, consumables and equipment used by the authors, while alternatives can be used in most of the cases, as long as the overall instructions and conditions are kept.
Materials
Isopropanol, 99.5%, molecular biology gradeThermo Fisher ScientificCatalog #327272500
Ethanol 70%AGA-Álcool e Géneros Alimentares, SA
TE buffer (Tris-EDTA) 1xSigma aldrich.com
LysozymeSigma aldrich.com
Genomic DNA purification kitFERMENTAS Inc.
RNAse, DNAse-freeRoche
Chloroform (Trichloromethane)Bio Basic Inc.Catalog #CC3000.SIZE.1L
NaCl (1.2M)Thermo Fisher
Absolute Ethanol
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
microalgae
Protocol materials
TE buffer (Tris-EDTA) 1xSigma aldrich.com
RNAse, DNAse-freeRoche
Genomic DNA purification kitFERMENTAS Inc.
Chloroform (Trichloromethane)Bio Basic Inc.Catalog #CC3000.SIZE.1L
Isopropanol, 99.5%, molecular biology gradeThermo Fisher ScientificCatalog #327272500
NaCl (1.2M)Thermo Fisher
Absolute Ethanol
Ethanol 70%AGA-Álcool e Géneros Alimentares, SA
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
LysozymeSigma aldrich.com
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.
DNA extraction
2m
Add 200 µL of TE buffer (Tris-EDTA) 1xSigma aldrich.com to the microalgae and vortex.
2m
Add powdered LysozymeSigma aldrich.com (enough to cover the surface of the liquid); incubate at 37ºC for 1 h in a thermoblock.
1h 2m
Add 400 µL of lysis solution from the Genomic DNA purification kitFERMENTAS Inc. and mix by inversion; incubate at 65ºC for 10 min.
12m
Let the samples reach Room Temperature (RT).
15m
Add 10 µL of RNAse, DNAse-freeRoche and incubate at 37ºC for 1 h (the concentration of RNAse and the incubation time are dependent on the type of biomass and the amount of RNA present).
1h 2m
Add 600 µL Chloroform (Trichloromethane)Bio Basic Inc.Catalog #CC3000.SIZE.1L ; mix by inversion.
2m
Centrifuge at maximum speed for 10 min at RT.
10m
Carefully, transfer the aqueous phase to a new tube (do not touch the interface or transfer any of the organic phase that is on the bottom).
1m
Add 1 volume of Isopropanol, 99.5%, molecular biology gradeThermo Fisher ScientificCatalog #327272500 ; mix by inversion for at least 5 times.
2m
Incubate at 4ºC for at least 30 min.
40m
Centrifuge at maximum speed for 30 min at RT.
35m
Discard the supernatant and resuspend in 100 µL NaCl (1.2M)Thermo Fisher
1m
Add 2.5 volumes of Absolute Ethanol (-20ºC); mix by inversion.
2m
Incubate at -20ºC for 30 min (or overnight).
30m
Centrifuge at maximum speed for 30 min at RT.
35m
Discard the supernatant and wash with 200 µL Ethanol 70%AGA-Álcool e Géneros Alimentares, SA , trying to detach the pellet as much as possible by beating the tube on the bench.
5m
Centrifuge at maximum speed for 5 min at RT.
6m
Discard the supernatant very well (let it dry – invert the tube into an absorbent paper for better efficiency). Resuspend in TE buffer (Tris-EDTA) 1xSigma aldrich.com (normally in 50 µL).
5m
Aliquot the samples (≈15 µl) for quantity and 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.
2m
gDNA quantity and quality check
1h 35m 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.
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 (microalgae )
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 (microalgae ). 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 (microalgae ), ratios around 1.9-2.0 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 (microalgae ), ratios around 2.0 can be reached; 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 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 45 min.
50m
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.