plantbiomethodsPrinciple
CTAB is a cationic detergent. In a buffer with high salt (1.4 M NaCl) it breaks open membranes and binds proteins and many polysaccharides, while nucleic acids stay in solution. EDTA chelates the magnesium that nucleases need. A chloroform extraction then removes the detergent–protein complexes into the organic phase, and isopropanol precipitates the DNA from the cleaned aqueous phase.
The method descends from Murray and Thompson (1980) and was adapted for small amounts of fresh tissue by Doyle and Doyle (1987). Its main strengths are cost and tolerance of difficult tissue. Its main weakness is that co-purifying polyphenols and polysaccharides can inhibit downstream enzymes if they are not controlled, which is what most of the critical steps below are about.
Safety
Chloroform is toxic and a suspected carcinogen, and it passes through nitrile gloves within minutes. Handle it only in a fume hood and change gloves immediately after contact. β-Mercaptoethanol is toxic and has a strong stench; add it to the buffer in the hood. Liquid nitrogen causes cold burns and can displace oxygen in small rooms; use a face shield and insulated gloves. Dispose of chloroform waste as halogenated solvent. Your institution's safety rules take precedence over this page.
Materials
Reagents
| Reagent | Notes |
|---|---|
| CTAB (hexadecyltrimethylammonium bromide) | Molecular biology grade |
| NaCl, 5 M stock | Filter or autoclave |
| Tris-HCl pH 8.0, 1 M stock | |
| EDTA pH 8.0, 0.5 M stock | |
| PVP-40 (polyvinylpyrrolidone), optional | For polyphenol-rich species |
| β-Mercaptoethanol | Added fresh, in the fume hood |
| Chloroform : isoamyl alcohol, 24 : 1 | Store in a dark bottle |
| Isopropanol | Keep at −20 °C |
| Ethanol, 70% (v/v) | Freshly made |
| TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) | Or nuclease-free water |
| RNase A, DNase-free (10 mg/mL stock) | |
| Liquid nitrogen |
Recipe: 2× CTAB extraction buffer (100 mL)
| Component | Final | Amount for 100 mL |
|---|---|---|
| CTAB | 2% (w/v) | 2 g |
| NaCl (5 M) | 1.4 M | 28 mL |
| Tris-HCl pH 8.0 (1 M) | 100 mM | 10 mL |
| EDTA pH 8.0 (0.5 M) | 20 mM | 4 mL |
| PVP-40 (optional) | 1% (w/v) | 1 g |
| Water | to 100 mL | |
| β-Mercaptoethanol, just before use | 0.2% (v/v) | 2 µL per mL used |
Warm to 60 °C and stir to dissolve the CTAB. Store without β-mercaptoethanol at room temperature for several months; CTAB precipitates in the cold, so rewarm before use.
Equipment
- Water bath or heat block at 65 °C
- Microcentrifuge reaching 12,000–16,000 × g, ideally refrigerated
- Mortar and pestle, or micropestles, or a bead mill with steel beads
- 1.5 or 2 mL tubes; fume hood; spectrophotometer; gel electrophoresis
Procedure
- Pre-warm the CTAB buffer to 65 °C. In the fume hood, add β-mercaptoethanol to the volume you need (2 µL per mL).Allow 600 µL per sample plus one spare.
- Harvest 50–100 mg of young leaf tissue into a labelled tube and freeze it immediately in liquid nitrogen.Young, expanding leaves give the highest yield and the least polysaccharide. Frozen tissue can be stored at −80 °C.
- CriticalGrind the tissue to a fine powder without letting it thaw.Pre-chill the pestle or bead holder in liquid nitrogen. Incomplete grinding is the most common cause of low yield; thawing before buffer is added releases nucleases and polyphenol oxidases.
- Add 600 µL of warm CTAB buffer to the frozen powder and vortex at once until no clumps remain.
- 30–60 minIncubate at 65 °C, inverting the tubes every 10 minutes.
- Cool to room temperature for 2–3 minutes. In the fume hood, add 600 µL chloroform : isoamyl alcohol (24 : 1) and mix by inversion for 5 minutes.Mix by inversion rather than vortexing if you need high-molecular-weight DNA for long-read sequencing.
- Centrifuge at 12,000 × g for 10 minutes at room temperature.
- CriticalTransfer the upper aqueous phase to a fresh tube without disturbing the interphase.Expect about 450–500 µL. Leave 50 µL behind rather than take interphase.
- For polysaccharide- or polyphenol-rich tissue, repeat steps 6–8 once with fresh chloroform : isoamyl alcohol.
- Add 0.7 volumes of ice-cold isopropanol (about 350 µL for 500 µL) and mix gently by inversion.White strands of DNA often become visible.
- Pause pointIncubate at −20 °C for at least 30 minutes.You can leave samples overnight at this step.
- Centrifuge at 12,000–16,000 × g for 10 minutes, at 4 °C if possible. Pour off the supernatant.
- Wash the pellet with 500 µL of 70% ethanol, centrifuge for 5 minutes, and discard the ethanol. Repeat the wash once.
- Remove the last drops with a pipette and air-dry the open tube for 5–10 minutes.Stop as soon as the pellet turns translucent. Over-dried DNA is slow to dissolve.
- Dissolve the pellet in 50 µL TE containing RNase A at 20 µg/mL and incubate at 37 °C for 30 minutes.Pellets that resist dissolving can be warmed to 55 °C for 10 minutes.
- Check quality and quantity: measure absorbance and run 2–5 µL on a 0.8% agarose gel.Store at 4 °C for use within weeks, or at −20 °C long term.
Expected results
| Measure | Target | What it tells you |
|---|---|---|
| A260/A280 | 1.8–2.0 | Lower values suggest protein or phenol contamination |
| A260/A230 | ≥ 1.8, ideally 2.0–2.2 | Low values flag polysaccharides, polyphenols or residual CTAB |
| Agarose gel | One sharp band above 20 kb | A low smear means degradation; a bright band at the bottom means RNA |
Yield varies widely between species and tissues; expect several to a few tens of micrograms from 100 mg of young leaf. For quantification before sequencing, use a dye-based fluorometric assay rather than absorbance, which overestimates DNA in the presence of RNA and contaminants.
Troubleshooting
| Problem | Likely cause | What to change |
|---|---|---|
| Low yield | Incomplete grinding; old or woody tissue; too much tissue for the buffer volume | Grind finer; use young leaves; keep to 100 mg per 600 µL |
| Brown extract or pellet; low A260/A230 | Oxidised polyphenols | Add 1% PVP-40; raise β-mercaptoethanol to 0.5%; work quickly from frozen powder |
| Gelatinous, viscous pellet | Co-precipitated polysaccharides | Repeat the chloroform step; use less tissue; re-precipitate with 0.7 vol isopropanol after adding NaCl to 1 M |
| Smear on the gel | Tissue thawed before buffer; nuclease activity | Keep tissue frozen until buffer is added; vortex into buffer immediately |
| PCR fails despite good yield | Inhibitors carried over | Dilute template 1:10; re-precipitate; add a second ethanol wash |
| Pellet will not dissolve | Over-drying | Warm to 55 °C for 10 min, then leave at 4 °C overnight |
References
- Doyle, J. J., & Doyle, J. L. (1987). A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin, 19, 11–15.
- Murray, M. G., & Thompson, W. F. (1980). Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 8(19), 4321–4325. doi:10.1093/nar/8.19.4321
Change log
1.0 (25 October 2026). First version.
How to cite this protocol
Kumar A. CTAB extraction of genomic DNA from plant leaf tissue. plantbiomethods.com, version 1.0, updated 25 October 2026. https://www.plantbiomethods.com/protocols/ctab-genomic-dna-extraction.html