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.
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)
2 ml centrifuge tubes
TE Buffer (10 mM Tris, pH 8, 1 mM EDTA)
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 µl 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 µl 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 µl 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 µl TE buffer (10 mM Tris, pH 8, 1 mM EDTA). The pellet may need warming in order to dissolve.
The use of silica spin columns can yield higher quality DNA. For an optional protocol, click here.