Mar 28, 2025

Public workspaceHigh molecular weight DNA extraction from algae : SoCK (Sorbitol + CTAB + KAc)

DOI
https://dx.doi.org/10.17504/protocols.io.j8nlkdbzwg5r/v1
High molecular weight DNA extraction from algae : SoCK (Sorbitol + CTAB + KAc)
  • Benoît acherie1,
  • Karine Labadie2
  • 1Genoscope/CEA;
  • 2Genoscope
  • Benoit
  • HPM/Genoscope
Benoît Vacherie
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DOI: https://dx.doi.org/10.17504/protocols.io.j8nlkdbzwg5r/v1
Protocol Citation: Benoît acherie, Karine Labadie 2025. High molecular weight DNA extraction from algae : SoCK (Sorbitol + CTAB + KAc). protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkdbzwg5r/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: March 25, 2025
Last Modified: March 28, 2025
Protocol Integer ID: 124949
Keywords: extraction, DNA, algae, HMW, Long read sequencing, high molecular weight, high molecular weight dna extraction protocol from algae, high molecular weight dna extraction from algae, algae with sorbitol, high molecular weight dna extraction protocol, high molecular weight dna extraction, dna extraction, algae, flash frozen algal fragment, types of algae, frozen algal fragment, conventional extraction with ctab, algal fragment, conventional extraction, extraction, dna, purification, sorbitol, purification with potassium acetate, molecular weight dna, weight dna, high molecular weight
Abstract
High Molecular Weight DNA extraction protocol from algae for Long Read sequencing.
Extraction is performed from flash frozen algal fragments stored at -80° C.
The protocol is adapted for an extraction of 1 g of algal fragment.
The protocol is based on washing the algae with Sorbitol, followed by a conventional extraction with CTAB and then a purification with Potassium acetate coupled with chloroform.
This protocol is specifically adapted to all types of algae (green, red or brown).
Guidelines
Use only wide bore tips.
Work in a chemical hood when using 2-mercaptoethanol.
Allow the DNA to resuspend for at least of 24 hours before proceeding with QC.

Materials
Reagents : ReagentTris HCl Buffer 1M Solution, Sterile pH 8.0Bio Basic Inc.Catalog #SD8127.SIZE.450ml
ReagentEDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
ReagentCTAB (Hexadecyltrimethylamm onium bromide) BBI BiotechCatalog #CB0108-100g
ReagentSodium chlorideP212121
ReagentPEG-8000
ReagentSorbitolP212121
ReagentPolyvinylpyrrolidone (PVP-40)Bio Basic Inc.Catalog #PB0436.SIZE.250g Reagent1X TE buffer (10 mM Tris-HCl pH 8.0 1 mM EDTA) Reagent70% Ethanol
ReagentSodium acetateP212121
Reagent2-Propanol (IsoPropanol)Bio Basic Inc.Catalog #PC8601.SIZE.4L ReagentChloroform Isoamyl alcohol 24:1Merck
ReagentPotassium Acetate
ReagentEthanol absolute
ReagentRNaseAThermo FisherCatalog #12091021 Reagent2-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6250 ReagentLiquid Nitrogen Equipment : ReagentEppendorf ThermoMixer Cpipette.comCatalog #2231000667 ReagentMortar and Pestels
ReagentVortex Mixer
Chemical fume hood

Protocol materials
ReagentLiquid Nitrogen
Reagent2-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
ReagentEthanol absolute
ReagentPotassium Acetate
ReagentChloroform Isoamyl alcohol 24:1Merck
ReagentRNaseAThermo FisherCatalog #12091021
Reagent2-Propanol (IsoPropanol)Bio Basic Inc.Catalog #PC8601.SIZE.4L
ReagentSodium acetateP212121
Reagent70% Ethanol
Reagent1X TE buffer (10 mM Tris-HCl pH 8.0 1 mM EDTA)
ReagentEppendorf ThermoMixer Cpipette.comCatalog #2231000667
ReagentVortex Mixer
ReagentPolyvinylpyrrolidone (PVP-40)Bio Basic Inc.Catalog #PB0436.SIZE.250g
ReagentEDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
ReagentTris HCl Buffer 1M Solution, Sterile pH 8.0Bio Basic Inc.Catalog #SD8127.SIZE.450ml
ReagentCTAB (Hexadecyltrimethylamm onium bromide) BBI BiotechCatalog #CB0108-100g
ReagentMortar and Pestels
ReagentSodium chlorideP212121
ReagentPEG-8000
ReagentSorbitolP212121
Troubleshooting
Preparation of reagents
Sorbitol pre-wash buffer : the buffer can be stored for 6 months at 4°C


ReagentsFinal Concentration
Tris HCl ph8100 mM
EDTA5 mM
Sorbitol700 mM
PVP 401%
H2OQsp 100 ml

Lysis Buffer

ReagentsFinal Concentration
Tris HCl ph8100 mM
EDTA20 mM
CTAB2 %
NaCl1.4 M
PEG 80001 %
H2OQsp 20 ml

Sorbitol Wash
39m
Add 20 ml of Sorbitol pre-wash Buffer in a 50 ml tube.
Amount20 mL Sorbitol buffer

Put a mortar in ice.
Cool the mortar and pestle with liquid nitrogen until the bubbling stops.
ReagentLiquid Nitrogen

10m
Grind 1g of frozen sample to a fine powder (approx. 2 min), without adding liquid nitrogen.
Amount1 g Duration00:02:00

2m
Transfer powder to sorbitol pre-wash buffer
Add 20 µl of 2-Mercaptoethanol and vortex 5 s
Reagent2-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6250

Centrifuge 5 minutes with low deceleration
Centrifigation5000 x g, Room temperature, 00:05:00 , Acc 9 / Dec 7

5m
Carrefully remove the supernatant without resuspending the pellet.
2m
Perform a second sorbitol wash as above (step 3 + 7 + 8 +9)
20m
DNA extraction and purification
5h 30m
Resuspend the pellet with 20 ml of Lysis buffer.
Add 50 µl of 2-Mercaptoethanol and 40 µl of RNaseA (100mg/ml) and vortex 5 s
Reagent2-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6250 ReagentRNaseAThermo FisherCatalog #12091021

Incubate 1h at 65°C with low agitation (300 rpm)
Duration01:00:00 Temperature65 °C

1h
Centrifuge 10 minutes with low deceleration
Centrifigation8500 x g, 4°C, 00:10:00 , Acc 9 / Dec 7

10m
Gently transfer the supernatant to a new 50 ml tube.
Add :
- 0.1 vol of absolute ethanol ReagentEthanol absolute
- 0.25 vol of KAc 3M ReagentPotassium Acetate
- 1 vol of chloroform isoamyl alcohol ReagentChloroform Isoamyl alcohol 24:1Merck

Vortex 5 sec and incubate 20 min at -20°C
Duration00:20:00 Temperature-20 °C

20m
Centrifuge 20 minutes with low deceleration
Centrifigation8500 x g, 4°C, 00:20:00 , Acc 9 / Dec 7
20m
Gently transfer the aqueous phase to a new 50 ml tube.
Add :
- 0.8 vol of isopropanol Reagent2-Propanol (IsoPropanol)Bio Basic Inc.Catalog #PC8601.SIZE.4L
- 0.1 vol of NaAc 3M ReagentSodium acetateP212121
- 0.2% of 2-MercaptoethanolReagent2-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6250

mix gently by inversion 10 times and incubate 1h at -20°C
Duration01:00:00 Temperature-20 °C

1h
Centrifuge 20 minutes with low deceleration
Centrifigation8500 x g, 4°C, 00:20:00 , Acc 9 / Dec 7
20m
Carefully discard the supernatant, gently resuspend the pellet with 1 ml of cold 70% ethanol.
Reagent70% Ethanol

Transfer the resuspended DNA to a 1.5 ml DNA LoBind tube.

Centrifuge 10 minutes with low deceleration
Centrifigation5000 x g, 4°C, 00:10:00 , Acc 9 / Dec 7
10m
Carefully discard the supernatant.
Air dry the pellet at RT for about 10 minutes. Duration00:10:00

10m
Resuspend the pellet with 50-100 µl of 1X TE buffer.
Reagent1X TE buffer (10 mM Tris-HCl pH 8.0 1 mM EDTA)

Allow the DNA to resuspend for 2 hours at 55°C or ON at RT.
Duration02:00:00 Temperature55 °C

2h
Store the DNA at 4°C.
Sample quality control
Quantify your sample with a Qubit DNA HS assay.
Check the purity of the sample with a Nanodrop (measurements of 260/280 and 260/230 absorbance ratios).
Estimate the molecular weight of the sample with a Tapestation and/or a Femto pulse
Depending on the DNA concentrations and DNA length profiles, deplete short DNA molecules using SRE size selection kits (SRE XS, SRE or SRE XL kits).
Results
QC results obtained on different species. (3 green algae and 3 red algae)