Feb 13, 2026
CTAB DNA extraction of high polysaccharide samples
- 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. CTAB DNA extraction of high polysaccharide samples. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrypb2gmk/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 26, 2026
Last Modified: February 13, 2026
Protocol Integer ID: 241310
Keywords: HMW-DNA, Biodiversity Genomics Europe, BGE, CTAB, CTAB DNA extraction, high polysaccharide samples, sorbitol wash, ctab hmw dna extraction, ctab hmw dna extraction for ground sample, hmw dna, ctab, high polysaccharide sample, polysaccharide, cetyltrimethyl ammonium bromide, extraction, dna, optional sorbitol wash
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 protocol describes a CTAB HMW DNA extraction for ground samples high in polysaccharides, including an optional sorbitol wash.
Acronyms:
CTAB = cetyltrimethyl ammonium bromide
HMW = high molecular weight
Attachments
Materials
- D-sorbitol (only if performing sorbitol wash)
- PVP40
- Tris-HCl pH 8
- EDTA pH 8
- Nuclease-free water/Milli-Q water
- BME (β-Mercaptoethanol)
- CTAB
- NaCl
- PEG-8000
- Proteinase K
- Pipettes, pipette tips
- optional: wide-bore p1000 pipette tips if you wish to transfer the DNA extract to a 1.5 ml LoBind tube
- 50 ml tubes
- 2 ml LoBind tubes if working with smaller non-plant samples
- Vortexer
- Centrifuge
- Thermomixer
- Chloroform:isoamylalcohol (24:1)
- Isopropanol
- 99 % ethanol
- Tube rotator (optional)
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 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.
If the tissue sample is ground in cryogenic conditions, take care to follow appropriate safety measures.
Before start
- NB: this protocol only works for ground samples.
- For plants, prepare 20 mL of CTAB lysis buffer in a 50 mL tube, which will accommodate a maximum of 1 gram plant tissue. If low yield is expected, prepare 10 ml extra CTAB lysis buffer per sample.
- For smaller non-plant samples (<50 mg) smaller volumes can be used. Use 2 ml tubes for lysis with a total of 1 ml CTAB lysis buffer.
- Sorbitol wash is usually not employed for non-plant samples, but can be beneficial when high amounts of polysaccharides are expected (e.g., crustaceans, whole insects, etc.)
Reagents used:
CTAB, a cationic detergent, constitutes a long hydrophobic hydrocarbon chain and a hydrophilic head. It forms micelles in water because of the amphipathic nature. During DNA extraction, under aqueous condition, CTAB comes in contact with the biological membrane, captures the lipids, and results in the release of nucleus, which is devoid of membrane.
CTAB at low ionic strength (<0.5M): precipitates nucleic acids and acidic polysaccharides.
CTAB at high ionic strength (>1.4M): binds sugars, denatures proteins and inhibits enzymes.
Buffer Recipes:
Sorbitol wash buffer
| Reagents for Sorbitol wash buffer | Final conc. | Stock | For 1 ml | For 30 ml | For 500 ml | |
| D-Sorbitol (182.17 g/mol) | 0.35 M | powder | 0.064 g | 1.91 g | 31.88 g | |
| PVP40 | 1 % | 10 % | 100 µl | 3 ml | 50 ml/5 g | |
| Tris-HCl pH8 | 100 mM | 1M | 100 µl | 3 ml | 50 ml | |
| EDTA pH 8 | 5 mM | 0.5 M | 10 µl | 300 µl | 5 ml | |
| NFW/MQW | - | - | to 1 ml | to 30 ml | to 500 ml | |
| To be added individually: | ||||||
| BME | 4 % | 100 % | 40 µl | 1200 µl | NA |
Recipe for Sorbitol wash buffer
CTAB lysis buffer
| Reagents for CTAB lysis buffer | Final conc. | Stock | For 1 ml | For 20 ml | For 500 ml | |
| Tris-HCl pH8 | 100 mM | 1M | 100 µl | 2 ml | 50 ml | |
| EDTA pH 8 | 25 mM | 0.5 M | 50 µl | 1 ml | 25 ml | |
| CTAB | 4 % | - | 0.04 g | 0.8 g | 20 g | |
| NaCl (58.44 g/mol) | 2 M | - | 0.12 g | 2.34 g | 58.44 g | |
| PEG-8000 | 3 % | 30 % | 100 µl | 2 ml | 50 ml/15 g | |
| PVP40 | 1 % | 10 % | 100 µl | 2 ml | 50 ml/5 g | |
| NFW/MQW | - | - | to 1 ml | to 20 ml | to 500 ml | |
| To be added individually: | ||||||
| BME | 2 % | 100 % | 20 µl | 400 µl | NA | |
| Proteinase K | 0.4 mg/ml | 20 mg/ml | 20 µl | 400 µl | NA |
Recipe for CTAB lysis buffer
Sorbitol Wash
20m
Sorbitol wash is done according to Sorbitol washing complex homogenate for improved DNA extractions by Jones and Schwessinger with minor changes.
Transfer 30 ml sorbitol wash buffer to a 50 ml tube, add 1200 μl BME and mix.
2m
Transfer 0.2-1 gram of frozen ground sample tissue to the tube and immediately shake, invert and vortex to mix thoroughly.
2m
Centrifuge at 4,700 x g for 00:05:00 at RT.
5m
Carefully decant the supernatant. Remove as much of the wash solution as possible without losing the pellet (Supernatant should appear slightly cloudy or discolored light yellow or brown.)
1m
If the supernatant was turbid, viscous or had dark discoloration, repeat the sorbitol wash.
9m
Proceed to a DNA extraction method of choice by adding lysis buffer to the pellet.
1m
CTAB Extraction
4h
Add BME and Proteinase K to individual tubes with CTAB lysis buffer and preheat to 65 °C.
3m
Transfer the pre-warmed CTAB lysis buffer from previous step to the sorbitol washed pellet and vortex tube 5 sec. (or as much as needed to get a homogenous suspension).
1m
Incubate min. 01:00:00 at 65 °C with agitation (800 rpm).
1h
Let the tube cool to RT.
5m
Add ~1 volume of chloroform:isoamylalcohol (24:1) and vortex/shake to make sure phases mix properly.
1m
Centrifuge at 20 °C, 00:10:00 , 4,700 x g with acceleration (9) and deceleration (6).
Note
Centrifugation creates heat and we want to maintain temperature without going below 15 °C.
The slower deceleration will create a more even/horizontal interphase and make clean transfer easier (only applies to swinging bucket rotors).
15m
Gently transfer the aqueous phase to a new tube.
Note
If the DNA is hard to transfer after the chloroform spin (big clumps near the interphase), consider breaking up the DNA more by transferring most of it to a new tube and either pipetting or vortexing before doing a second chloroform rinse. If DNA is too HMW it will be hard to pellet and you might lose most of it anyway.
1m
For higher yield, add 10 ml pre-warmed CTAB lysis buffer to the organic phase and repeat step 7 and transfer the aqueous phase into the same tube as before.
15m
For higher purity, repeat steps 6-8 (if you did step 9, use <1x volume).
15m
Add 0.6-0.7 volume of RT isopropanol and gently mix by inversion 10 times.
2m
Incubate tube at RT for min. 00:15:00 to allow for better precipitation.
15m
Centrifuge at 20 °C, 00:30:00 , 4,700 x g.
30m
Carefully remove the supernatant by gentle decanting without disturbing the pellet.
Note
For 2 ml tubes, remove the supernatant with a pipette instead.
1m
Wash with freshly prepared 70 % EtOH. Gently swirl and invert the tube before leaving the pellet to soak for 00:15:00 at RT or use a tube rotator at 10-20 rpm.
17m
Centrifuge 20 °C, 00:10:00 , 4,700 x g.
10m
Carefully decant the supernatant without disturbing the pellet.
Note
If transfer to 1.5 ml tube is desirable, you can add 1 ml freshly prepared 70 % EtOH and transfer the pellet to the smaller tube using a wide-bore p1000 tip.
1m
Repeat steps 15-17 at least once.
Note
If precipitating with isopropanol, consider a 3rd wash to further remove excess salt.
28m
Perform a quick spin to gather the residual ethanol at the bottom of the tube and carefully remove with a P20 tip. Repeat if needed.
5m
Let pellet air dry for 5-15 min00:10:00 at room temperature, taking care not to over dry.
15m
Add resuspension buffer (preferred Elution Buffer/lowTE/TE) and leave at RT with gentle mixing overnight to resuspend. No pipetting at this time! Alternatively, leave at 4°C/RT for 3-4 days to increase homogeneity before performing quality control.
Short incubation at 50 °C may promote faster resuspension, especially if pellet has been slightly overdried.
It can take several weeks for some samples to rehydrate properly. The higher the concentration and integrity, the longer it takes for the sample to become homogenous.
If needed, the resuspended DNA can be RNase treated and precipitated again.
Note
For HiFi libraries, the DNA can be sheared and cleaned with Ampure beads (0.5X) instead of precipitation.
Protocol references
Sorbitol wash is done according to https://www.protocols.io/view/sorbitol-washing-complex-homogenate-for-improved-d evow1875pr2/v1 with minor changes