DNA Extraction from Plant Samples using CTAB Method
A comprehensive, published-grade molecular biology case study detailing isolation of high-purity plant genomic DNA via Cetyltrimethylammonium Bromide (CTAB) lysis, followed by Nanodrop spectrophotometry, PCR amplification, and agarose gel electrophoresis.
Deoxyribonucleic Acid (DNAThe molecule carrying genetic instructions for development and functioning of living organisms.) extraction from plant tissues poses unique biochemical challenges due to rigid cellulose cell walls, abundant polyphenols, and complex polysaccharides. The CTABCetyltrimethylammonium bromide, a cationic detergent that solubilizes membranes and forms complexes with DNA. method is the gold-standard protocol designed to bind nucleic acids while precipitating polysaccharides and preventing polyphenolic oxidation.
Key Downstream Applications
Marker-assisted selection (MAS) and cultivar verification.
Species identification using rbcL and matK chloroplast genes.
High-molecular-weight DNA prep for Illumina and Nanopore WGS.
qPCR target validation and transgene verification.
01 Plant Sample Collection
Young, tender leaves are selected to minimize polysaccharide accumulation and polyphenol oxidation.
Collect 100–200 mg of fresh young leaf tissue. Wash thoroughly with double-distilled water to remove epiphytic microbes and dust.
Old Leaves: High polyphenols cause brown DNA pellets. Always use young expanding leaves.
Use sterile tweezers and gloved hands to prevent human DNA contamination.
02 Sample Preparation
Tissue is flash-frozen in liquid nitrogen (-196°C) to immediately arrest nuclease activity.
03 Mechanical Grinding
Grinding leaf tissue in a pre-chilled mortar and pestle breaks the rigid cellulose cell wall.
04 CTAB Lysis Buffer Preparation
The buffer components solubilize cell membranes while preserving DNA integrity.
| Reagent | Concentration | Scientific Purpose |
|---|---|---|
| CTAB | 2% (w/v) | Solubilizes cell membrane lipids and precipitates polysaccharides. |
| NaCl | 1.4 M | High salt concentration maintains DNA solubility while CTAB binds. |
| Tris-HCl (pH 8.0) | 100 mM | Buffers pH to prevent acid hydrolysis of DNA. |
| EDTA (pH 8.0) | 20 mM | Chelates divalent cations ($Mg^{2+}$) to inhibit DNase enzymes. |
| β-Mercaptoethanol | 0.2% (v/v) | Reduces disulfide bonds and inactivates polyphenols. |
05 Cell Lysis & Buffer Addition
Pre-warmed CTAB buffer (65°C) is added to the ground leaf powder to disrupt lipid bilayers.
06 Water Bath Incubation
Samples are incubated at 65°C for 30–60 minutes with periodic gentle inversion.
07 Phase Separation (Chloroform:Isoamyl Alcohol 24:1)
Organic solvent extraction separates proteins, lipids, and cellular debris from aqueous nucleic acids.
08 Centrifugation
Centrifuge at 12,000 rpm for 10 minutes at 4°C to resolve phase separation.
09 DNA Precipitation (Isopropanol)
Addition of ice-cold isopropanol (0.6 volumes) precipitates genomic DNA into visible white threads.
10 DNA Pellet Washing (70% Ethanol)
Wash pellet twice with 70% ethanol to remove residual salts and organic solvents.
11 Air Drying Pellet
Invert microcentrifuge tube under sterile laminar airflow for 15 minutes to dry residual ethanol.
12 DNA Dissolution
Resuspend purified DNA pellet in 50 μL 1X TE Buffer (10 mM Tris-HCl, 1 mM EDTA) or nuclease-free water.
13 DNA Quality Analysis (Nanodrop Spectrophotometry)
Absorbance ratios assess DNA purity and concentration.
| Ratio / Parameter | Pure Range | Meaning of Deviations |
|---|---|---|
| $A_{260}/A_{280}$ | 1.8 – 2.0 | < 1.8 indicates protein/phenol contamination; > 2.0 indicates RNA. |
| $A_{260}/A_{230}$ | 2.0 – 2.2 | < 2.0 indicates salt, carbohydrate, or CTAB contamination. |
Step 14: Polymerase Chain Reaction (PCR) Amplification
In vitro enzymatic amplification of target DNA loci using specific primers and Taq polymerase.
3-Stage Thermal Cycling
95°C (30s)
55–60°C (30s)
72°C (60s)
PCR Components Table
| Component | Volume (25 μL Reaction) | Final Concentration |
|---|---|---|
| 2X PCR Master Mix (Taq + dNTPs) | 12.5 μL | 1X |
| Forward Primer (10 μM) | 1.0 μL | 0.4 μM |
| Reverse Primer (10 μM) | 1.0 μL | 0.4 μM |
| Template DNA (~50 ng/μL) | 2.0 μL | ~100 ng |
| Nuclease-Free Water | 8.5 μL | N/A |
Step 15: Agarose Gel Electrophoresis
DNA fragments migrate through a 1% agarose matrix toward the positive anode ($+$) based on molecular weight.
Interactive Gel Result Comparison (Clean Band vs. Smeared/Failed)
Drag handle left/right to compare clean high-molecular-weight band vs. degraded smeared DNA.
Graph & Result Interpretation
Nanodrop Spectral Curve
Peak at 260 nm without secondary 230 nm shoulder indicates clean CTAB removal.
Gel Lane Analysis
Single sharp band >10 kb proves minimal shearing during mechanical grinding.
Biosafety & Laboratory Precautions
Complete 20-Stage Workflow Summary
Fresh leaf sampling through mortar powdering under liquid nitrogen.
65°C incubation, Chloroform extraction, and high-speed centrifugation.
Precipitation, Nanodrop verification, PCR amplification, and gel documentation.
References & Academic Protocols
- Doyle, J. J., & Doyle, J. L. (1987). A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin, 19, 11-15.
- Sambrook, J., & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor Laboratory Press.