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Cetyltrimethyl Ammonium Bromide (CTAB) DNA Miniprep for Plant DNA Isolation
Joseph D. Clarke

Cold Spring Harb Protoc 2009; doi: 10.1101/pdb.prot5177

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Protocol

Cetyltrimethyl Ammonium Bromide (CTAB) DNA Miniprep for

Plant DNA Isolation
Joseph D. Clarke

INTRODUCTION
The presence of polysaccharides in a DNA preparation can inhibit use of techniques such as poly-
merase chain reaction (PCR). Cetyltrimethyl ammonium bromide (CTAB) is a surfactant useful for iso-
lation of DNA from tissues containing high amounts of polysaccharides. Under the high-salt conditions
used in this protocol, the CTAB binds the polysaccharides, removing them from the solution. When
used with Arabidopsis, this procedure yields quite pure DNA that is suitable for thermal asymmetric
interlaced PCR (TAIL-PCR).

RELATED INFORMATION
This protocol is adapted from that of Doyle and Doyle (1990). Other methods for plant DNA isolation
can be found in Dellaporta Miniprep for Plant DNA Isolation (Weigel and Glazebrook 2009a),
Quick Miniprep for Plant DNA Isolation (Weigel and Glazebrook 2009b), and An Expedient and
Versatile Protocol for Extracting High-Quality DNA from Plant Leaves (Vallejos 2007).

MATERIALS
CAUTIONS AND RECIPES: Please see Appendices for appropriate handling of materials marked with <!>, and
recipes for reagents marked with <R>.

Reagents
<!>Chloroform:isoamyl alcohol (96:4)
<R>CTAB DNA extraction buffer
Ethanol (70%), cold
<!>Isopropanol
<!>Phenol:chloroform:isoamyl alcohol (48:48:4)
Plant tissue, ~100 mg per sample
TE buffer (optional; see Step 12)

Equipment
<!>Liquid nitrogen
Microcentrifuge
Microcentrifuge tubes
Micropestles or mortars and pestles (see Step 2)
Micropipettor with tips
Vortex mixer
Water bath preset to 65ºC

Adapted from Arabidopsis: A Laboratory Manual (eds. Weigel and

Glazebrook). CSHL Press, Cold Spring Harbor, NY, USA, 2002.
Cite as: Cold Spring Harb. Protoc.; 2009; doi:10.1101/pdb.prot5177 www.cshprotocols.org

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METHOD

1. Collect ~100 mg of plant tissue in a standard microcentrifuge tube. Store the tissue at −80ºC
before use.
2. Grind the tissue in liquid nitrogen using a plastic pestle in the tube itself, or with a mortar and
pestle.
If desired, the ground tissue may be stored at −80°C.

3. Preheat the CTAB extraction buffer to 65ºC in a water bath and estimate the volume of ground
plant tissue in each sample.
4. Add 1 volume of hot CTAB extraction buffer to each tissue sample and incubate for 10-30 min
at 65ºC.
5. Add 1 volume of phenol:chloroform:isoamyl alcohol (48:48:4) to the tube and vortex thoroughly.

6. Centrifuge in a microcentrifuge at maximum speed for 2 min.

7. Transfer the aqueous layer to a clean microcentrifuge tube and repeat the extraction (Steps 3-5)
using chloroform:isoamyl alcohol (96:4).
8. Add 0.7 volume of isopropanol, mix by inversion, and incubate for 10 min at room temperature.

9. Pellet the DNA in a microcentrifuge at maximum speed for 15 min at room temperature.

10. Discard the supernatant and wash the pellet with 0.5 mL of cold 70% ethanol. Centrifuge briefly
to secure the pellet.
11. Carefully discard the supernatant and dry the pellet under vacuum or in air.

12. Dissolve the DNA in 50 µL of water or TE buffer.

REFERENCES
Doyle, J.J. and Doyle, J.L. 1990. Isolation of plant DNA from fresh
tissue. Focus 12: 13–15.
Vallejos, C.E. 2007. An expedient and versatile protocol for extracting
high-quality DNA from plant leaves. Cold Spring Harb. Protoc. doi:
10.1101/pdb.prot4765.
Weigel, D. and Glazebrook, J. 2009a. Dellaporta miniprep for plant
DNA isolation. Cold Spring Harb. Protoc. (this issue). doi: 10.1101/
pdb.prot5178.
Weigel, D. and Glazebrook, J. 2009b. Quick miniprep for plant DNA
isolation. Cold Spring Harb. Protoc. (this issue). doi: 10.1101/
pdb.prot5179.

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