Jan 09, 2026
HMW DNA extraction from red algae for Long Read Sequencing
- 1Genoscope/CEA
- Benoit
- HPM/Genoscope
Protocol Citation: Benoît acherie, Karine Labadie 2026. HMW DNA extraction from red algae for Long Read Sequencing . protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwn577vmk/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: January 07, 2026
Last Modified: January 09, 2026
Protocol Integer ID: 237186
Keywords: rhodophyta, algae, DNA extraction, HMW DNA extraction, sarkosyl, PVP, hmw dna extraction from red algae, hmw dna extraction, high molecular weight dna extraction, dna extraction, extraction of dna, red algae, read sequencing, long read sequencing, extraction, dna, high molecular weight, purification, mercaptoethanol,
Abstract
This protocol describes High Molecular Weight DNA extraction for Long Read Sequencing.
This protocol was adapted from Ramakrishnan et al. (2017; doi: 10.1007/s13205-017-0992-2), with some modifications.
This protocol is designed for the extraction of DNA from 200 mg of material that has been flash-frozen in liquid nitrogen and stored at –80 °C.
The procedure involves cell lysis in a Sarkosyl-based buffer supplemented with PVP and β-mercaptoethanol, followed by purification using potassium acetate and chloroform:isoamyl alcohol.
Guidelines
Use only wide bore tips.
Work in a chemical hood when using béta-mercaptoéthanol and chloroform.
Allow the DNA to resuspend for at least of 24 hours before proceeding with QC.
Protocol materials
Liquid Nitrogen
RNAse A (DNAse free) 100mg/ml QiagenCatalog #19101
70% ethanol
DNA LoBind Tubes 1.5 mlEppendorfCatalog #022431021
Beta-mercaptoethanolCatalog #97622-10X1ML
Isopropanol
Sodium acetate, 3MFisher ScientificCatalog #J61928
Chloroform:Isoamyl Alcohol [24:1]Merck MilliporeSigma (Sigma-Aldrich)Catalog #25666
Ethanol absolute
Potassium Acetate
Buffer TE 1x
Preparation of reagents
30m
Extraction Buffer : Prepare 10 mL of extraction buffer (per 200g of material) in a 50 mL tube
| Reagents | Final concentration | |
| Tris-HCl ph8 | 100 mM | |
| EDTA | 20 mM | |
| NaCl | 1.5 M | |
| Sarkosyl | 1% | |
| PVP40 | 2% | |
| H2O | QSP 10 ml |
30m
DNA Extraction
1h 26m
Add 10 ml of extraction buffer to a 50 ml tube.
10 mL Extraction buffer
1m
Put a mortar in ice.
Cool the mortar and pestle with liquid nitrogen until the bubbling stops.
Liquid Nitrogen
5m
Grind 200 mg of frozen sample to a fine powder (approx. 2 min).
200 mg Sample
2m
Transfer the powder to the extraction buffer
1m
Add 20 µl of Beta-mercaptoethanol to the extraction buffer and vortex 5 sec.
Beta-mercaptoethanolCatalog #97622-10X1ML
1m
Incubate at room temperature for 1 h with agitation (300 rpm).
1h
Centrifuge with low deceleration
8500 x g, 4°C, 00:10:00 , Acc 9 / Dec 6
15m
Transfer the supernatant to a new 50 ml tube.
1m
DNA Purification
2h 4m
Add to the supernatant :
0.1 volume (1 ml) of absolute ethanol Ethanol absolute
0.25 volume (2.5 ml) of 3M potassium acetate Potassium Acetate
1 volume (10 ml) of chloroform isoamyl alcohol Chloroform:Isoamyl Alcohol [24:1]Merck MilliporeSigma (Sigma-Aldrich)Catalog #25666
Vortex 2x 5 sec
1m
Incubate for 20 min at -20°C
20m
Centrifuge with low deceleration
8500 x g, 4°C, 00:20:00 , Acc 9 / Dec 6
25m
Gently transfer the aqueous phase to a new 50 ml tube.
1m
Add 2µl of RNase A (100mg/ml)
RNAse A (DNAse free) 100mg/ml QiagenCatalog #19101
1m
Incubate for 30 min at 37°C
30m
Add 1 volume (10 ml) of chloroform isoamyl alcohol Chloroform:Isoamyl Alcohol [24:1]Merck MilliporeSigma (Sigma-Aldrich)Catalog #25666
Vortex 2x 5 sec
1m
Incubate for 20 min at -20°C
20m
Centrifuge with low deceleration
8500 x g, 4°C, 00:20:00 , Acc 9 / Dec 6
25m
Gently transfer the aqueous phase to a new 50 ml tube.
DNA précipitation
4h 2m
Beta-mercaptoethanolCatalog #97622-10X1ML Add to the supernatant :
0.8 volume (≈ 8 ml) of isopropanol Isopropanol
0.1 volume (≈ 1 ml) of 3M sodium acetate Sodium acetate, 3MFisher ScientificCatalog #J61928
0.2% (≈ 2 µl) of β-mercaptoethanol Beta-mercaptoethanolCatalog #97622-10X1ML
1m
Homogenise by inversion and incubate for 1 hour at -20°C.
1h
Centrifuge with low deceleration
8500 x g, 4°C, 00:20:00 , Acc 9 / Dec 6
25m
Carefully discard the supernatant, gently resuspend the pellet with 1 ml of cold 70% ethanol.
70% ethanol
5m
Transfer the resuspended DNA to a 1.5 ml DNA LoBind tube.
DNA LoBind Tubes 1.5 mlEppendorfCatalog #022431021
5m
Centrifuge with low deceleration
5000 x g, 4°C, 00:10:00 , Acc 9 / Dec 6
15m
Carefully discard the supernatant.
Air dry the pellet at RT for about 5-10 minutes.
10m
Resuspend the pellet in 50-100 µl of 1X TE buffer.
Buffer TE 1x
1m
Allow the DNA to resuspend for 2 hours at 55°C or ON at RT.
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.