Feb 13, 2026
Phenol/Chloroform DNA Extraction Protocol with SDS Lysis Buffer
- Mai-Britt Mosbech1
- 1Department of Immunology, Genetics and Pathology, SciLifeLab Genomics NGI, Uppsala Genome Center, Uppsala University, Sweden
- Biodiversity Genomics Europe
Protocol Citation: Mai-Britt Mosbech 2026. Phenol/Chloroform DNA Extraction Protocol with SDS Lysis Buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq1ej5vk5/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 04, 2026
Last Modified: February 13, 2026
Protocol Integer ID: 242618
Keywords: BGE, hmw dna extraction, phenol chloroform extraction, sds lysis buffer, pci, dna extraction, Biodiversity Genomics Europe, chloroform hmw dna extraction protocol, chloroform dna extraction protocol with sds lysis buffer, chloroform dna extraction protocol, hmw dna, sds lysis buffer, extraction protocol, phenol, extraction, dna, protocol
Funders Acknowledgements:
Biodiversity Genomics Europe receives funding from the European Union's Horizon Europe Research and Innovation Action
Grant ID: 101059492
Disclaimer
The duration times presented are estimations only.
Abstract
This is a Phenol/Chloroform HMW DNA Extraction Protocol with SDS Lysis Buffer.
Acronyms:
HMW = High molecular weight
SDS = Sodium Dodecyl Sulfate
Attachments
Materials
- N-lauryl sarcosine
- SDS
- Tris pH 7-9
- EDTA
- Proteinase K
- BME (β-Mercaptoethanol) (optional)
- Nuclease-free water/Milli-Q water
- PVP10 (optional)
- PVP40 (optional)
- PVP360 (optional)
- NaCl
- TE 100x (only if using TE-buffer)
- 2 ml Eppendorf safe-lock tube
- Puregen Proteinase K (2.5 mg/ml final concentration)
- Scalpel + other material to slice tissue, e.g. weighing boat
- Puregen Proteinase K
- Thermoshaker
- Centrifuge
- RNase A
- 2 ml MaXtract High Density phase lock tube, Qiagen
- Pipettes + standard pipette tips and p1000 wide-bore tip
- Minicentrifuge
- Phenol:Chloroform:Isoamyl Alcohol (25:24:1)
- Chloroform:Isoamyl Alcohol (24:1)
- Tube rotator
- 99% ethanol
Troubleshooting
Safety warnings
The operator must wear a lab coat, powder-free nitrile gloves and work in a fume hood to perform the laboratory procedures in this protocol. In addition, chloroform-resistant gloves must be worn, and phenol and chloroform waste bottles should be stored in the fume hood.
Waste needs to be collected in a suitable container and disposed of in accordance with local regulations.
Liquid waste needs to be collected in a suitable container and disposed of in accordance with local regulations.
See best practices, thawing and handling instructions, and safety instructions for all the kit reagents, as well as instructions and safety data sheets for other reagents used in this protocol before starting.
Ethics statement
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Before start
- Proteinase K and RNase A are added individually to samples and should not be added to the lysis buffer stock.
- BME (β-Mercaptoethanol) and PVPs (Polyvinylpyrrolidones) can be added to remove polyphenolic compounds and prevent them from binding to the DNA. It has been indicated that BME and the different PVPs bind different types of polyphenols; however, this has not been explicitly confirmed. You can choose to add one or more of these if you suspect phenolic content in your input material.
- The high salt/low ethanol precipitation will precipitate small amounts of polysaccharides, which would otherwise co-precipitate with the DNA.
- For input material high in polysaccharides, we recommend using CTAB (Cetrimonium bromide) lysis buffer instead; however, this does not work with the MaXtract tubes due to the high salt content in the lysis buffer. It is possible to buy phase-gel tubes for high salt samples; however, we have not tested these.
- Aim for 25-200 mg tissue depending on tissue type and expected DNA yield. For PacBio HiFi, we would optimally like 20-30 µg of DNA in 200 µl corresponding to 100-150 ng/µl. If you expect a much lower yield, consider eluting in a lower volume as purity measurement by absorbance becomes more unreliable with concentrations below 10 ng/µl.
- Start at step 8 when cleaning already extracted DNA; bring each sample to 405 µl with sample buffer.
Buffer Recipes:
SDS lysis buffer
| Reagents for SDS lysis buffer | Stock | Final conc. | Vol. for 400 µl | Vol. for 10 ml | |
| N-lauryl Sarcosine | 10% | 2% | 80 µl | 2 ml | |
| SDS | 20% | 0.5% | 10 µl | 250 µl | |
| Tris pH 7-9 | 1 M | 50 mM | 20 µl | 500 µl | |
| EDTA | 0.5 M | 10 mM | 8 µl | 200 µl | |
| Proteinase K (add fresh) | 20 mg/ml | 2.5 mg/ml | 50 µl | Add individually | |
| BME (optional, add fresh) | 100% | 1 % | 4 µl | 100 µl | |
| PVP10 (optional) | 10% | 1 % | 40 µl | 1 ml | |
| PVP40 (optional) | 10% | 1 % | 40 µl | 1 ml | |
| PVP360 (optional) | 5% | 1 % | 80 µl | 2 ml | |
| MQ-H2O | NA | NA | Up to 400 µl | Up to 10 ml |
Recipe for SDS lysis buffer
Note
NaCl will be added after the lysis step.
Buffer A
| Reagents for Buffer A | Stock | Final conc. | Vol. for 1 ml | |
| Tris pH 7-9 | 1 M | 10 mM | 10 µl | |
| EDTA | 0.5 M | 2 mM | 4 µl | |
| NaCl | 5 M | 1 M | 200 µl | |
| MQ-H2O | NA | NA | 786 µl |
Recipe for Buffer A
LowTE-buffer
| Reagents for LowTE-buffer | Stock | Final conc. | Vol. for 50 ml | |
| Tris pH 7-9 | 1 M | 10 mM | 500 µl | |
| EDTA | 0.5 M | 0.1 mM | 10 µl | |
| MQ-H2O | NA | NA | 49.4 ml |
Recipe for LowTE-buffer
TE-buffer
| Reagents for TE buffer | Stock | Final conc. | Vol. for 400 µl | |
| TE 100x | Tris 1 M/EDTA 100 mM | Tris 10 mM/EDTA 1 mM | 500 µl | |
| MQ-H2O | NA | NA | 49.5 ml |
Recipe for TE-buffer
PCI Extraction protocol using SDS lysis buffer
4h 45m
Label 2 ml Eppendorf safe-lock tube and fill it with 350 μl of working SDS lysis buffer.
1m
Slice frozen tissue sample with scalpel and transfer sliced tissue into tube with SDS lysis buffer.
Note
Work fast to get the tissue into the SDS lysis buffer before thawing too much. If tissue is very soft, an Eppendorf pestle can be used to break up the tissue in the SDS lysis buffer by squishing it against the sides of the tube. NO rotating pestle. Never use TissueRuptor together with SDS lysis buffer.
2m
Add 50 μl Puregen Proteinase K (2.5 mg/ml final concentration) per tube.
Incubate sample at 50°C for at least00:30:00 . Shake at 900-1400 rpm for the first 10-15 minutes to improve lysis and reduce fragment size for higher purity. Reduce to 300-450 rpm after the 15 min and incubate until all tissue has lysed.
Note
If the tissue is well sliced, complete lysis can occur in 30 minutes at 50°C. In 2 hours, lysis occurs in most of tubes unless they contained high amount of tissue.
2h
Lower temperature to 37°C and add 5 µl RNase A (10 mg/ml, final conc. 120 µg/ml).
1m
Incubate at 37°C, 300 rpm for 00:30:00 .
30m
Label the 2 ml MaXtract High Density phase lock tube from Qiagen. Spin it for 20-30 seconds at 12-16,000 x g.
1m
Shortly spin tube with lysate to remove buffer from cap.
Transfer lysate with a p1000 wide-bore tip set to 500 μl to phase lock tube.
Note
If a fat layer is present, try to avoid this while pipetting.
1m
Add 100 μl of 5 M NaCl (1 M final concentration).
In the fume hood, add 400 μl of Phenol:Chloroform:Isoamyl Alcohol (25:24:1).
Note
Before the actual distribution of phenol:chloroform, fill the tip and empty it 2-3 times to avoid dripping.
1m
Close tubes firmly and mix the organic and aqueous phase thoroughly to form a transiently homogeneous suspension. Do not vortex. Tube rotation is okay (e.g., 20-30 rpm for 00:10:00 ).
11m
Spin for 00:10:00 at 16,000 x g.
Note
The original protocol says 1 min, but HMW-DNA may need longer spin time to separate phases properly. It may be hard to retrieve the HMW-DNA closest to the gel as this will follow when you try to pipette the viscous DNA liquid.
11m
Carefully add 400 μl of Chloroform:Isoamyl Alcohol (24:1) on top of the aqueous phase and mix thoroughly without breaking the gel-seal to the organic phase. Do not vortex! Tube rotator (20 rpm, 00:10:00 ) seems to be okay for the samples tested at our lab.
Note
As previously, fill and empty the tip several times before distributing to prevent dropping of chloroform.
11m
Spin for 00:10:00 at 16,000 x g.
11m
Transfer up to 450 μl of the aqueous phase to a new, labeled 2 ml tube and make a note of the transferred volume.
1m
(Optional) Add as much buffer A to the phase lock tube as needed, in order for final sample volume to reach 650 µl and mix thoroughly without breaking the gel-seal to the organic phase. Spin for 2-5 minutes at 12,000 x g and transfer approximately the same amount of the aqueous phase as you added in this step to the previously transferred volume for a total of maximum 650 µl.
10m
Add 0.3x vol of 99% ethanol and invert tube 20 times to mix.
Note
This high-salt, low-ethanol mixture precipitates polysaccharides while gDNA remains in solution.
2m
Spin tube at maximum speed (at least 10,000 x g) for 00:15:00 .
15m
Carefully transfer the supernatant to a new labeled 2 ml tube without disturbing the polysaccharide pellet.
Note
No visible pellet may be seen at this step but always transfer as if a pellet is present.
1m
Add 1.7x volume of 99% ethanol (according to transferred sample volume before addition of ethanol in step 19) and invert tube 20 times to mix.
2m
Spin tube at maximum speed (at least 10,000 x g) for00:30:00 .
31m
Carefully remove supernatant, do not disturb the pellet.
1m
Add 1000 µl of freshly prepared 70% ethanol to remove the excess salt; try to avoid dislodging the pellet.
1m
Spin tube at maximum speed (at least 10 g) for 00:05:00 .
6m
Carefully remove supernatant, do not disturb the pellet.
1m
Repeat steps 24-26.
8m
Perform a quick spin to gather the residual ethanol at the bottom of the tube and carefully remove with a P20 tip.
Let pellet air dry for 00:05:00 at room temperature, taking care not to over dry.
5m
Add 100-200µl resuspension buffer (preferred Elution Buffer/lowTE/TE) and leave at RT with gentle mixing overnight to resuspend. Alternatively, leave at 4°C/RT for 3-4 days to increase homogeneity before performing quality control.
Note
It can take several weeks for some samples to re-hydrate properly. The higher the concentration and integrity, the longer it takes for the sample to become homogeneous.