CTAB Protocol for Isolating DNA from Plant Tissues

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

The following protocols for isolating clean plant DNA, both start with a traditional approach using a cetyltrimethylammonium bromide (CTAB) buffer. At that point they diverge, the first protocol makes use of phenol and chloroform, and the second protocol uses a reverse solid phase extraction (i.e., capturing contaminants on a solid phase). Methods using phenol and chloroform are preferred for the isolation of high molecular weight DNA, however both chemicals are considered hazardous. Phenol can cause very serious chemical burns while chloroform is a known carcinogen. The CTAB method using solid phase extraction, avoids phenol and chloroform and is best used for assays where smaller DNA fragment sizes are acceptable.

Skip ahead to the protocols:

Plant DNA Isolation using Phenol/Chloroform Extraction

Plant DNA Isolation using Reverse Solid Phase Extraction

 

Isolation Chemistry

As mentioned, polysaccharides and polyphenols are problematic when isolating DNA from plant tissues. CTAB buffers are effective at removing polysaccharides and polyphenols from plant DNA preparations. CTAB (also called hexadecyltrimethylammonium bromide) is a cationic detergent that facilitates the separation of polysaccharides during purification while additives, such as polyvinylpyrrolidone, aid in inactivating polyphenols. CTAB based extraction buffers are widely used when purifying DNA from plant tissues. The hazard with traditional CTAB protocols is the protein component of plant lysates is usually removed using phenol and chloroform. These two solvents are generally considered hazardous. The solid phase protocol listed below is an alternative. CTAB is more than a surfactant and its properties can be used in several ways to purify DNA. One option for purifying DNA using CTAB exploits the different solubilities of polysaccharides and DNA in CTAB depending upon the concentration of sodium chloride. At higher salt concentrations (1.4 M), polysaccharides are insoluble, while at lower concentrations (600 mM) DNA is insoluble. Consequently, adjusting salt concentration in lysates with CTAB, polysaccharides and DNA can be differentially precipitated. Most methods use CTAB to remove polysaccharides, followed by protein removal and DNA separation using precipitation or spin columns.

Plant cells contain phenolic compounds, such as catechol, that are catalyzed by polyphenol oxidase to o-quinones. The o-quinones in turn can alkylate and inactivate proteins. Polyphenol oxidases are found in plastids (i.e., chloroplasts) while catechol is found in vacuoles. When plant cells and tissues are disrupted, the enzyme and substrate mix and generate the reactive o-quinones (which is associated with browning of damaged leaves and fruit). Therefore, homogenizing plant tissue yields reactive molecules that can potentially interfere with subsequent manipulation of the DNA. To avoid the production of o-quinones, phenolic precursors are captured by polyvinylpyrrolidone (PVP) that is present in the homogenization buffer. PVP binds strongly with aromatic compounds, such as catechol and subsequent polyphenols, and prevents the formation of reactive o-quinones. CTAB-based protocols tend to work very well, but with one significant disadvantage phenol/chloroform extractions are routinely used to separate protein from the DNA. As chloroform is carcinogenic, many institutions frown upon its use. Furthermore, phenol can cause serious chemical burns. The traditional protocol will be covered, as well as an alternative protocol that uses solid phase extraction. The solid phase extraction is the basis of the Synergy™ Plant DNA Extraction Kit.

Plant DNA Isolation using Phenol/Chloroform Extraction

This method is best for isolating high molecular weight DNA. Caution is needed when working with liquid nitrogen, chloroform, and phenol. Consult your organization’s safety guidelines when working with these hazardous materials.

Materials

• CTAB buffer: 2% cetyl trimethylammonium bromide, 1% polyvinylpyrrolidone, 100 mM Tris-HCl, 1.4 M NaCl, 20 mM EDTA, or CTAB Extraction Buffer
• Polypropylene tubes (don’t use polycarbonate tubes with phenol and chloroform)
• Centrifuge (at least 14,000 x g)
• RNase A Solution (10 mg/ml in water, DNase-free)
• Isopropanol
• 70% Ethanol
• 2 ml polypropylene centrifuge tubes
• Centrifugal Vacuum Concentrator (e.g., SpeedVac)
• TE Buffer (10 mM Tris, pH 8, 1 mM EDTA)
• Phenol/Chloroform/Isoamyl Alcohol (25:24:1 ratio) stored under TE buffer, pH 8

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.

1. Transfer the ground plant tissue to a polypropylene tube.
2. For every 100 mg of homogenized tissue add 500 µl of CTAB Buffer. Mix and thoroughly vortex.
3. Place the tube in a 60°C water bath for 30 minutes.
4. Centrifuge the homogenate for 5 minutes at 14,000 x g.
5. Transfer supernatant to a new tube.
6. Add 5 µl of RNase A solution and incubate at 37°C for 20 minutes.
7. Add an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1).
8. Vortex for 5 seconds then centrifuge the sample for 1 minute at 14,000 x g to separate the phases.
9. Transfer the aqueous upper phase to a new tube. Repeat this extraction until the upper phase is clear.
10. Transfer the upper aqueous phase to a new tube.
11. Add 0.7 volume cold isopropanol and incubate at -20°C for 15 minutes to precipitate the DNA.
12. Centrifuge the sample at 14,000 x g for 10 minutes.
13. Decant the supernatant without disturbing the pellet and subsequently wash with 500 µl ice cold 70% ethanol.
14. Decant the ethanol. Remove the residual ethanol by drying in a SpeedVac.
15. Dry the pellet long enough to remove alcohol, but without completely drying the DNA.
16. Dissolve the DNA pellet in 20 µl TE buffer (10 mM Tris, pH 8, 1 mM EDTA). The pellet may need to be warmed, in order to dissolve.

Optional protocol:

Protocol for higher quality DNA, using silica spin columns to further purify the DNA.

Plant DNA Isolation using Reverse Solid Phase Extraction (i.e., Synergy™ protocol)

This method eliminates the use of phenol and chloroform but requires the use of a bead beater. Bead beaters such as the Tissuelyser, GenoGrinder®, MiniG®, FastPrep, and small dental amalgamators will work. Bead beating shears DNA, thus DNA isolated is typically 2-7 Kb, sufficient for most Next-Generation Sequencing (NGS) and Polymerase Chain Reaction (PCR).

Materials

• Synergy™ 2.0 Plant DNA Extraction kit (SYNP 02-100-02)
  • Synergy™ Homogenization Tubes
  • Plant Homogenization Buffer
  • RNase A solution
  • Silica Spin Columns (Product # SSC 100-01)
• Bead Beater (any homogenizer that holds 2 ml tubes should work)
• Isopropanol
• 70% Ethanol, stored at -20°C
• TE Buffer, pH 8/ Molecular Biology Grade Water
• Microcentrifuge
• Microfuge tubes, 1.7 ml, snap cap

Method

1. Place up to 50 mg of plant tissue in Synergy™ homogenization tube, then add 500 µl Plant Homogenization Buffer. Leafy tissue and young roots homogenize well. Seeds require pre-grinding to meal.
2. Homogenize via bead beating (approximately 5K oscillations/min) for two minutes. The lysate should appear milky. If the tube contains foam, repeat the bead beating.
3. Once the sample is milky, centrifuge for 5 minutes at 15,000 x g, to pellet contaminants and debris. The homogenization matrix will both disrupt the sample and then capture the contaminants. This will be obvious with leaf samples as chlorophyll will be captured by the solid phase. Pelleting the solid phase will yield a clear lysate. A small hydrophobic layer can sometimes be observed on the top of the lysate.
4. Transfer the cleared lysate to a new tube and add 5 µl RNase A solution. Incubate for 15 minutes at 37°C. At this point the lysate can be used for PCR but it might require some dilution if the plant has high levels of PCR inhibitors.
5. Add 0.7 volumes of isopropanol and incubate at -20°C for 15 minutes.
6. Transfer the solution to a silica spin column, with a collection tube and centrifuge at low speed to slowly push the DNA solution through the silica membrane.
7. Discard the flow through and place the column back into the collection tube.
8. Wash by adding 250 µl of ice cold 70% ethanol and centrifuge at 8000 x g.
9. Discard the flow through and repeat the wash.
10. Transfer the column to a clean collection tube.
11. Elute in 50- 100 µl of molecular biology grade water or TE buffer and centrifuge at 15,000 x g for 1 minute. The DNA can be stored at -20°C until needed.