CTAB Protocol for Isolating DNA from Plant Tissues

Isolating DNA from plant tissues can be very challenging as the biochemistry
between divergent plant species can be extreme. Unlike animal tissues where the
same tissue type from different species usually have similar characteristics,
plants can have variable levels of metabolites and structural biomolecules.
Polysaccharides and polyphenols are two classes of plant biomolecules that vary
widely between species and are very problematic when isolating DNA. Contaminating
polysaccharides and polyphenols can interfere with manipulations of DNA following
isolation.

Methods are available that effectively remove polysaccharides and polyphenols from
plant DNA preparations. The use of CTAB (cetyl trimethylammonium bromide), a
cationic detergent, facilitates the separation of polysaccharides during
purification while additives, such as polyvinylpyrrolidone, can aid in removing
polyphenols. CTAB based extraction buffers are widely used when purifying DNA from
plant tissues.

One option for purifying DNA using CTAB exploits that polysaccharides and DNA have
different solubilities in CTAB depending on the concentration of sodium chloride.
At higher salt concentrations, polysaccharides are insoluble, while at lower
concentrations DNA is insoluble. Consequently, by adjusting salt concentration in
lysates containing CTAB, polysaccharides and DNA can be differentially
precipitated.

Polyphenols are compounds that contain more than one phenolic ring (e.g., tannin),
a structure that binds very efficiently to DNA. They are naturally occurring in
plants, but are also generated when plants have tissue damage (browning). Upon the
homogenization of plant tissues, polyphenols are synthesized by liberated
polyphenol oxidase. The addition of polyvinyl pyrrolidone prevents the interaction
of DNA and phenolic rings by binding up the polyphenols.

CTAB-based protocols tend to work very well, but one significant disadvantage is
that chloroform extractions are routinely used to separate organic soluble
molecules from the DNA. As chloroform is carcinogenic, many institutions frown
upon its use. Thus, an alternative method that avoids chloroform has been
developed by OPS Diagnostics; it can be found on the Synergy� Plant DNA Extraction
Kit page.

Materials Needed

CTAB buffer: 2% cetyl trimethylammonium bromide, 1% polyvinyl pyrrolidone,100 mM

Tris-HCl, 1.4 M NaCl, 20 mM EDTA, or CTAB Extraction Buffer

Centrifuge (up to 14,000 x g)

Isopropanol

70% Ethanol

2 ml centrifuge tubes

SpeedVac

TE Buffer (10 mM Tris, pH 8, 1 mM EDTA)

Method

Plant samples can be prepared by cryogenically grinding tissue in a mortar and

pestle after chilling in liquid nitrogen. Freeze dried plants can be ground at room
temperature. In either case, a fine powder is best for extracting DNA.

For each 100 mg homogenized tissue use 500 �l of CTAB Extraction Buffer. Mix and
thoroughly vortex. Transfer the homogenate to a 60�C bath for 30 minutes.

Following the incubation period, centrifuge the homogenate for 5 minutes. at 14,000
x g.

Transfer supernatant to a new tube. Add 5 �l of RNase solution A and incubate at

32�C for 20 minutes

Add an equal volume of chloroform/isoamyl alcohol (24:1). Vortex for 5 seconds then
centrifuge the sample for 1 min. at 14,000 x g to separate the phases. Transfer the
aqueous upper phase to a new tube. Repeat this extraction until the upper phase is
clear.

Transfer the upper aqueous phase to a new tube. Precipitate the DNA by adding 0.7
volume cold isopropanol and incubate at -20�C for 15 minutes.

Centrifuge the sample at 14,000 x g for 10 minutes. Decant the supernatant without
disturbing the pellet and subsequently wash with 500 �l ice cold 70% ethanol.
Decant the ethanol. Remove residual ethanol by drying in a SpeedVac.

Dry the pellet long enough to remove alcohol, but without completely drying the
DNA. Dissolve DNA in 20 �l TE buffer (10 mM Tris, pH 8, 1 mM EDTA). The pellet may
need warming in order to dissolve.

Optional protocol:

The use of silica spin columns can yield higher quality DNA. For an optional
protocol, click here.