Apr 23, 2025

Public workspaceMicrobial DNA extraction from the brown seaweed Ascophyllum nodosum

DOI
dx.doi.org/10.17504/protocols.io.8epv52kx6v1b/v1
  • 1Biology of Marine Models (LBI2M, UMR 8227), Station Biologique de Roscoff, Sorbonne Université, CNRS
Coralie Rousseau
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DOI: dx.doi.org/10.17504/protocols.io.8epv52kx6v1b/v1
Protocol Citation: Coralie Rousseau, Sylvie Rousvoal, Catherine Leblanc, Simon M. Dittami 2025. Microbial DNA extraction from the brown seaweed Ascophyllum nodosum. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv52kx6v1b/v1
Manuscript citation:
Rousseau C. , Henry N., Rousvoal S., Tanguy G., Legeay E., Leblanc C., Dittami S.M. Comparison of short and long-read metabarcoding sequencing: challenge and solutions for plastid read removal and microbial
community exploration of seaweed samples, (2024) 10.22541/au.172675397.78191661/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: February 11, 2025
Last Modified: April 23, 2025
Protocol Integer ID: 119953
Keywords: DNA extraction, Brown macroalgae, Ascophyllum nodosum, Metabarcoding, Polysaccharides, Metagenomics, High molecular weight, Microbial DNA extraction, PCR inhibitors
Abstract
This protocol is the result of optimizing microbial DNA extraction from the brown alga Ascophyllum nodosum. This protocol includes three different DNA purification steps to remove polysaccharide contaminants and other potential PCR inhibitors. This protocol takes 1.5 days for 22 samples

✅ Short- and long-read sequencing
✅ High molecular weight is obtained (>40 kb)
✅ Sucessfully tested for metagenome sequencing (skip step 1, carry out step 2 in liquid nitrogen, and use 10 mg wet weight of sample in triplicate, and skip the bead beater)

This protocol has been successfully applied to other brown seaweeds:
✅ Sargassum
Materials
Consumables & Chemicals
Reagents / Buffers:
  • Acetone (100%)
  • CTAB 10% (Cetyltrimethylammonium bromide)
  • PVPP (Polyvinylpolypyrrolidone)
  • NaCl 5M (Sodium Chloride)
  • Tris HCL pH 8
  • EDTA pH 8
  • DIECA 0.1M (Sodium Diethyldithiocarbamate, ref. 228680, SigmaAldrich)
  • β-mercaptoethanol (ref. M31148, SigmaAldrich)
  • Chloroform/isoamyl alcohol (24:1)
  • Ethanol 100%
  • Ethanol 70% (with Biological grade water)
  • Sodium acetate solution 3M
  • Biological grade water (ref. 46-000-CV, Corning, USA)
  • NucleoSpin Plant II kit (MachereyNagel, Germany)
  • Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA)

Disposable materials:

  • 1.5 mL standart Eppendorf tubes (standard)
  • 2 mL standart Eppendorf tubes (standard)
  • 2 mL Safe-Lock Eppendorf tubes (ref. 0030120094)
  • Parafilm
  • Steel beads for TissueLyser

Lab Equipments:
  • Freeze-dryer (lyophilizer) (Qiagen, Hilden, Germany)
  • Mortar (for manual grinding)
  • Qiagen TissuLyser II (bead beater)
  • Vortex mixer
  • Centrifuges
  • Incubator
  • DynaMag‱-2 Magnet (ref. 12321D, ThermoFisher)
  • Manual pipettes
  • Dry ice
  • -80°C freezer
  • -20°C freezer
Sample prepration
Sample prepration
1m
1m
Dry matter in freeze-dryer at least Duration48:00:00

  • In sterile condition, grind sample by hand with sterile mortar and pestle until a very fine powder is obtained

Note
Clean with ethanol 70% mortar and pestle to avoid contaminations between samples

  • Take 10 mg of ground algal tissue, put into 2 mL Eppendorf tube with 1 steel bead
  • Put in Qiagen TissueLyser II bead beater (Duration00:01:00 , 30 Hz)
Sodium diethyldithiocarbamate (CTAB) Buffer preparation (for 22 samples)
Sodium diethyldithiocarbamate (CTAB) Buffer preparation (for 22 samples)
10m
10m
Prepare in a 50 mL Falcon tube:
ReagentInitial concentrationFinal concentrationVolume to put
CTAB 10%1024.6 mL
PVPP (g)10020.46 g
Tris-HCl pH 810001002.3 mL
NaCL 5M 51.46.44 mL
EDTA pH 8500200.92 mL
Sterile H208.74 mL
Total: 23 mL
Mix the preparation

Note
Keep CTAB buffer at 21°C or higher to prevent CTAB from precipitating

Use fresh buffer each extraction

Pipetting
Polyphenol Cleaning
Polyphenol Cleaning
16m
16m
  • AddAmount1 mL acetone (100%)
  • Vortex, 10 min
  • Centrifigation12800 x g, 21°C, 00:01:00
  • Remove acetone by pipetting
  • Repeat the previous steps
1m
Pipetting
Dry between Duration00:05:00 to Duration00:10:00 to remove any remaining acetone atTemperatureRoom temperature

15m
First DNA purification - Remove polysaccharides
First DNA purification - Remove polysaccharides
3h 30m
3h 30m
Remove bead from each tube by reversing the tube to make it roll away (bead added step 2)
In 2mL Eppendorf Safe-Lock Tubes:
  • add Amount1 mL of CTAB buffer to each sample
  • Add Amount35 µL of 0.1M DIECA (Sodium Diethyldithiocarbamate) + Amount10 µL of β-mercaptoethanol
  • Incubation during Duration01:00:00 at Temperature65 °C

  • Under Sorbonne, add Amount1 mL of chloroform/isoamyl (24:1, v/v)
Note
Add parafilm around each Eppendorf Safe-Lock Tube to avoid chloroform vapours
  • Vortex, 10 min
  • Centrifigation13200 rpm, 21°C, 00:30:00
  • Collect the upper phase (Amount750 µL ) in a new 2mL Eppendorf Safe-Lock Tube. Be careful to not collect the interphase
1h 30m
Pipetting
Toxic
To the upper phase:
  • Add Amount225 µL of ethanol 100% drop by drop and mix by pipette agitation inside the liquid
  • Add Amount975 µL of chloroform (100%)
Note
Add parafilm around each Eppendorf Safe-Lock Tube to avoid chloroform vapours
  • Vortex, 1 min
  • Centrifigation13200 rpm, 21°C, 00:20:00
  • Collect the upper phase (Amount550 µL ) in new 2mL Eppendorf Safe-Lock Tubes. Be careful to not collect the interphase
20m
Pipetting
Toxic
To the upper phase:
  • Add Amount110 µL of acetate sodium 3M solution
  • Add Amount1.1 mL of ethanol 100% to precipitate DNA (mixing by tube inversion)
  • Put samples in Temperature-80 °C during Duration01:00:00
Note
Prepare Amount50 mL of ethanol 70% and put the tube on ice


  • Centrifigation13200 rpm, 4°C, 00:30:00
  • Carefully remove liquid phase without disturbing DNA pellet 
1h 30m
Pipetting
Toxic
  • Add Amount500 µL of cold ethanol 70%
  • Vortex, 20s
  • Centrifigation13200 rpm, 4°C, 00:10:00
  • Carefully remove liquid phase without disturbing DNA pellet 

  • Repeat step 10 once
Note
Pellet could be translucide, white or yellow

10m
Pipetting
  • Spin down to collect remaining ethanol
  • Remove the upper phase by pipetting
  • Dry pellet during Duration00:05:00 at TemperatureRoom temperature to remove remaining ethanol
Add Amount50 µL of biological grade water and mix by pipetting 10 times

Note
STOPPING POINT Temperature-20 °C


Second DNA purification (with Nucleospin Plant Kit II)
Second DNA purification (with Nucleospin Plant Kit II)
13m
13m

Note
All of the following steps are processed in sterile conditions and performed according to the manufacturer’s instructions (except for step 17)

Place Buffer PE at Temperature65 °C

  • Add Amount400 µL of biological grade water to the previously extracted DNA and mix by pipetting 10 times
  • Add Amount400 µL in purple Nucleospin column 
  • Centrifigation11000 x g, 21°C, 00:02:00
  • Keep the flow-through which contains the DNA
  • Add Amount450 µL of Buffer PC
  • Mix by pipetting 5 times
2m
Pipetting
  • Add Amount450 µL in green NucleoSpin Column
  • Centrifigation11000 x g, 21°C, 00:01:00
  • Discard the flow-through.
  • Add the remainder in green NucleoSpin Column 
  • Centrifigation11000 x g, 21°C, 00:01:00
  • Discard the flow-through.
2m
Pipetting
  • Add Amount400 µL of buffer PW1 to the green column
  • Centrifigation11000 x g, 21°C, 00:01:00
  • Discard the flow-through.
1m
Pipetting
  • Add Amount700 µL of buffer PW2 to the green column
  • Centrifigation11000 x g, 21°C, 00:01:00
  • Discard the flow-through

  • Repeat the previous step

  • Add Amount200 µL of buffer PW2 to the green column
  • Centrifigation11000 x g, 21°C, 00:01:00
  • Discard the flow-through
2m
Pipetting
  • Put the green column on a clean 1.5 mL Eppendorf 
  • Elution process: Add Amount25 µL of Buffer PE on the green column membrane 
  • Incubation at Temperature65 °C during Duration00:05:00
  • Centrifigation11000 x g, 21°C, 00:01:00
  • Keep the flow-thought which contains the DNA

  • Repeat the elution process

Note
STOPPING POINT Temperature-20 °C


6m
Centrifigation
Thrid DNA purification (with Protocol Agencourt AMPure XP)
Thrid DNA purification (with Protocol Agencourt AMPure XP)
40m 30s
40m 30s

Note
All the following steps are processed in sterile conditions and performed according to the manufacturer’s instructions

1. Place the bottle of Agencourt AMPure XP at TemperatureRoom temperature Duration00:30:00 before use and shake it to resuspend any magnetic particles.

2. Prepare fresh 70% ethanol with biological grade water
30m
  • Add Amount90 µL of Agencourt AMPure XP (according to sample reaction volumes:  Volume AMPure = 1.8 x sample volume)
  • Mix by pipetting 10 times. Color must be homogenous
  • Incubation at TemperatureRoom temperature during Duration00:05:00
5m
Pipetting
On DynaMag‱-2 Magnet:
  • Put Eppendorf on magnetic plate during Duration00:02:00 . Wait for the solution to clear 
  • Removing slowly and discard the upper phase. Leave a few μL of supernatant behind, otherwise beads are drawn out with the supernatant.

  • Add Amount200 µL of ethanol 70% 
  • Incubation Duration00:00:30 at TemperatureRoom temperature
  • Slowly remove the supernatant by pipetting and discard
  • Repeat the previous steps
2m 30s
Remove the reaction plate from the magnet plate, dry the pellet to remove any trace of ethanol
Wait until the pellet is a little cracked
  • Add Amount50 µL of elution buffer PE (from kit NucleoSpin) 
  • Mix by pipetting 10 times 
  • Incubation during Duration00:02:00 at TemperatureRoom temperature  
2m
Pipetting
On DynaMag‱-2 Magnet:
  • Put Eppendorf on magnetic plate during Duration00:01:00
  • Transfert the DNA elution (upper phase) into a new 1.5 mL Eppendorf
1m

Note
The end.
Short-reads: Conservation at Temperature-20 °C
Long-reads: Conservation at Temperature-80 °C


Quality control
Quality control
DNA must be measured by Qubit Fluorometer (Thermo Fisher Scientific) or equivalent
Protocol references
Panova, M. et al. 2016. DNA Extraction Protocols for Whole-Genome Sequencing in Marine Organisms, pp. 13–44. In Bourlat, S.J., ed, Marine Genomics: Methods and Protocols. Springer, New York, NY.

Varela-Alvarez, E., Andreakis, N., Lago-Leston, A., Pearson, G.A., Serrao, E.A., Procaccini, G., Duarte, C.M., and Marba, N. 2006. Genomic data isolation from green and brown brown algae (Caulerpales and Fucales) for microsatellite library construction. J. Phycol. 42, 741–745.
Acknowledgements
We would like to thank Gwenn Tanguy (Genomer Platform, FR2424, Station Biologique de Roscoff, Sorbonne Université, CNRS) and Erwan Legeay (Genomer Platform, FR2424, Station Biologique de Roscoff, Sorbonne Université, CNRS, 29680 Roscoff, France & Adaptation and Diversity in the Marine Environment (UMR 7144), Station Biologique de Roscoff, Sorbonne Université, CNRS) for reviewing this protocol.