Mar 28, 2025

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

DOI
dx.doi.org/10.17504/protocols.io.j8nlkdbzwg5r/v1
  • 1Genoscope/CEA;
  • 2Genoscope
  • Benoit
  • HPM/Genoscope
Benoît Vacherie
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DOI: dx.doi.org/10.17504/protocols.io.j8nlkdbzwg5r/v1
Protocol Citation: Benoît Vacherie, 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
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-8000Contributed by users
ReagentSorbitolP212121
ReagentPolyvinylpyrrolidone (PVP-40)Bio Basic Inc.Catalog #PB0436.SIZE.250g Reagent1X TE buffer (10 mM Tris-HCl pH 8.0 1 mM EDTA)Contributed by users Reagent70% Ethanol Contributed by users
ReagentSodium acetateP212121
Reagent2-Propanol (IsoPropanol)Bio Basic Inc.Catalog #PC8601.SIZE.4L ReagentChloroform Isoamyl alcohol 24:1Merck
ReagentPotassium AcetateContributed by users
ReagentEthanol absoluteContributed by users
ReagentRNaseAThermo FisherCatalog #12091021 Reagent2-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6250 ReagentLiquid NitrogenContributed by users Equipment : ReagentEppendorf ThermoMixer Cpipette.comCatalog #2231000667 ReagentMortar and PestelsContributed by users
ReagentVortex MixerContributed by users
Chemical fume hood

Protocol materials
ReagentPotassium Acetate
Materials, Step 16
Reagent70% Ethanol
Materials, Step 23
ReagentVortex Mixer
Materials
ReagentPolyvinylpyrrolidone (PVP-40)Bio Basic Inc.Catalog #PB0436.SIZE.250g
Materials
Reagent2-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
In Materials and 3 steps
Reagent2-Propanol (IsoPropanol)Bio Basic Inc.Catalog #PC8601.SIZE.4L
Materials, Step 20
ReagentSodium acetateP212121
Materials, Step 20
Reagent1X TE buffer (10 mM Tris-HCl pH 8.0 1 mM EDTA)
Materials, Step 27
ReagentEppendorf ThermoMixer Cpipette.comCatalog #2231000667
Materials
ReagentLiquid Nitrogen
Materials, Step 4
ReagentEthanol absolute
Materials, Step 16
ReagentCTAB (Hexadecyltrimethylamm onium bromide) BBI BiotechCatalog #CB0108-100g
Materials
ReagentMortar and Pestels
Materials
ReagentSodium chlorideP212121
Materials
ReagentChloroform Isoamyl alcohol 24:1Merck
Materials, Step 16
ReagentRNaseAThermo FisherCatalog #12091021
Materials, Step 12
ReagentEDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
Materials
ReagentTris HCl Buffer 1M Solution, Sterile pH 8.0Bio Basic Inc.Catalog #SD8127.SIZE.450ml
Materials
ReagentPEG-8000
Materials
ReagentSorbitolP212121
Materials
Preparation of reagents
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
Sorbitol Wash
39m
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 NitrogenContributed by users

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
DNA extraction and purification
5h 30m
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 absoluteContributed by users
- 0.25 vol of KAc 3M ReagentPotassium AcetateContributed by users
- 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 Contributed by users

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)Contributed by users

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
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
Results
QC results obtained on different species. (3 green algae and 3 red algae)