Jan 28, 2026
CTAB–Chloroform DNA Extraction from Ethanol-Preserved Freshwater Phytobentos
- Pavla Urbankova1,
- Jan Štěpka1
- 1Faculty of Science, J. E. Purkyně University in Ústí nad Labem, Pasteurova 3632/15, 40096 Ústí nad Labem, Czech Republic
Protocol Citation: Pavla Urbankova, Jan Štěpka 2026. CTAB–Chloroform DNA Extraction from Ethanol-Preserved Freshwater Phytobentos. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpzxwdlzp/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
We use this protocol and it's working
Created: July 20, 2024
Last Modified: January 28, 2026
Protocol Integer ID: 103761
Keywords: metabarcoding, CTAB, DNA extraction, chloroform, isopropanol, chloroform dna extraction from ethanol, extraction of edna, chloroform dna extraction, freshwater phytobentos this protocol, single chloroform extraction, preserved freshwater phytobento, stream water sample, based extraction, preserved river, ethanol, ctab
Abstract
This protocol describes a CTAB-based extraction of eDNA from particulate material pelleted from an ethanol-preserved river/stream water sample. It uses a single chloroform extraction, isopropanol precipitation, two 70% ethanol washes, and elution in Qiagen EB buffer. The protocol is adapted from Strand et al. (2019) with modifications for a pellet-based workflow.
Guidelines
- Contamination prevention (recommended): Use filter tips, wear gloves, change gloves frequently, and decontaminate work surfaces with a bleach-based solution before and during the work.
- Pellet handling: DNA pellets may be small and can be nearly invisible to the naked eye. Use extra care when removing the isopropanol supernatant after precipitation and during the 70% ethanol wash steps to avoid pellet loss.
- Optional clarification step: If the recovered aqueous phase remains visibly turbid after chloroform separation, an additional chloroform extraction can be performed to improve phase clarity before precipitation.
Materials
Reagents
- CTAB buffer (pH 8.0): 100 mM
- Tris-HCl, 20 mM EDTA, 1.4 M NaCl, 2% (w/v) CTAB (no additives)
- Proteinase K (20 mg/mL)
- Chloroform, pure (p.a.)
- Isopropanol (p.a.)
- 70% ethanol (prepared from p.a. ethanol + autoclaved deionized water)
- Qiagen EB buffer (10 mM Tris-Cl, pH ~8.5)
Consumables
- 2.0 mL microcentrifuge tubes (standard snap-cap “Eppendorf” type)
- Filter tips
- Gloves
Equipment
- Microcentrifuge for 2.0 mL tubes (able to run at ~13,500 rpm / max speed)
- Temperature-controlled device (heat block, water bath, or incubator) capable of 65 °C
- Vortex mixer
- Vacuum concentrator (optional; alternative is heat block drying)
Troubleshooting
Safety warnings
- Chloroform is toxic and volatile: handle in a fume hood; dispose of organic waste properly.
- Ethanol and isopropanol are flammable: keep away from ignition sources.
- Bleach-based disinfectants are irritants/corrosive: wear gloves and avoid mixing with acids.
Before start
- Pre-cool isopropanol and 70% ethanol at −20 °C.
- Preheat a heat block, water bath, or a temperature-controlled incubator (suitable for microcentrifuge tubes) to 65 °C.
- Decontaminate the work surface and equipment and organise a clean workflow to minimise contamination.
Pellet preparation from ethanol-preserved sample (2 mL aliquot)
33m 30s
Resuspend particulates in the ethanol-preserved sample container by manual shaking until homogenized.
30s
Transfer 2 mL of homogenized ethanol mixture into a 2.0 mL microcentrifuge tube.
30s
Centrifuge at 13500 rpm, Room temperature, 00:20:00 (max speed).
21m
Carefully decant the supernatant without disturbing the pellet.
30s
Dry the pellet for 00:10:00 using either a vacuum concentrator or a heat block (open cap), ensuring ethanol is removed.
11m
CTAB lysis and Proteinase K digestion
2h 35m
Add 500 µL CTAB buffer (room temperature) to the dried pellet.
30s
Vortex continuously for 00:15:00 .
16m
Freeze at -20 °C until fully frozen (typically 01:00:00 ); samples may be stored at -20 °C Overnight and processed the next day.
1h 1m
Thaw at 65 °C for 00:15:00 .
16m
Add4 µL 20 µL Proteinase K (20 mg/mL ) and vortex briefly.
30s
Incubate at 65 °C for 01:00:00 .
1h 1m
Chloroform extraction
19m
Add 400 µL chloroform and mix gently with the pipette tip.
1m
Centrifuge at 13500 rpm, Room temperature, 00:15:00 .
16m
Carefully transfer ~300 µL of the upper aqueous phase to a new 2.0 or 1,5
mL tube using a fresh filter tip, avoiding the interphase.
2m
Isopropanol DNA Precipitation
33m
Add 400 µL ice-cold isopropanol (stored at -20 °C ) and mix by inverting the tube several times.
Incubate 00:15:00 at 4 °C .
16m
Centrifuge for 13500 rpm, Room temperature, 00:15:00 .
16m
Remove supernatant carefully without losing the pellet.
1m
Ethanol washes
15m
Add 500 µL ice-cold 70% ethanol (stored at -20 °C ) and briefly vortex.
1m
Centrifuge at 13500 rpm, Room temperature, 00:05:00 .
6m
Carefully remove supernatant.
30s
Repeat etanol wash once more .
7m 30s
Drying and elution
12m
Dry the pellet (open cap) for ~00:10:00 in a vacuum centrifuge or on a 65 °C heat block (avoid airborne contamination).
11m
Dissolve DNA pellet in 30 µL EB buffer (or sterile milliQ water), vortex and spin down. Let the DNA dissolve for at least 1 hour before further analysis (or store in fridge/freeze).
1m
Protocol references
Strand, D. A., Johnsen, S. I., Rusch, J. C., Agersnap, S., Larsen, W. B., Knudsen, S. W., Møller, P. R., & Vrålstad, T. (2019). Monitoring a Norwegian freshwater crayfish tragedy: eDNA snapshots of invasion, infection and extinction. Journal of Applied Ecology, 56(7), 1661–1673. https://doi.org/10.1111/1365-2664.13404