DNA Isolation from Plant Tissue Using CTAB Method — Complete Guide

Table of Contents

Introduction

Principle

Materials & Reagents

Step-by-Step Protocol

Results

Troubleshooting

Conclusion

FAQs

Introduction

Plant molecular biology fundamentally depends on the acquisition of pure DNA for subsequent applications, including PCR, sequencing, cloning, and genetic marker analysis. Plant tissues are frequently abundant in polysaccharides, phenolic compounds, and secondary metabolites that may compromise DNA integrity. The CTAB (Cetyltrimethylammonium Bromide) method is a very dependable and extensively utilized technique for isolating DNA from various plant species. This article offers a comprehensive discussion of the methodology, elucidation of the principle, and a structure for reproducible outcomes.

Principle

The CTAB method works on the principle of selective precipitation and removal of contaminants:

1. Cell Lysis: Plant cell walls are broken by grinding in liquid nitrogen, and membranes are lysed using CTAB buffer.
2. Role of CTAB: CTAB is a cationic detergent that forms complexes with polysaccharides and helps keep them soluble while allowing DNA to remain in solution.
3. Salt (NaCl): High concentration of NaCl prevents polysaccharide co-precipitation with DNA.
4. PVP (Polyvinylpyrrolidone): Binds polyphenols, which otherwise oxidize and bind irreversibly to nucleic acids.
5. β-mercaptoethanol: Reduces oxidative damage and deactivates nucleases.
6. Chloroform-Isoamyl Extraction: Removes proteins, lipids, and CTAB-polysaccharide complexes.
7. Precipitation: DNA is selectively precipitated by isopropanol and later washed with ethanol.
8. RNase Treatment: Removes RNA contamination, ensuring high purity DNA.

Materials & Reagents

Step-by-Step Protocol

Sample Preparation

1. Harvest ~100 mg of young, fresh leaf tissue.

2. Freeze with liquid nitrogen and grind to fine powder.

DNA Extraction

1. Add 700 µL preheated CTAB buffer (65 °C) to powdered tissue.

2. Incubate at 65 °C for 30 min, mixing gently.

3. Add equal volume chloroform:isoamyl alcohol (24:1), mix, and centrifuge at 12,000 × g for 10 min.

4. Transfer the aqueous phase carefully to a fresh tube.

RNA Removal

Add 2 µL RNase A and incubate at 37 °C for 20 min.

DNA Precipitation

1. Add 0.7 volume isopropanol, mix gently until DNA strands appear.

2. Incubate at room temp for 20 min (or −20 °C for higher yield).

3. Centrifuge at 12,000 × g for 10 min.

Washing

1. Wash pellet with 1 mL 70% ethanol.

2. Centrifuge and air-dry pellet for 5–10 min.

Resuspension

Dissolve DNA in 50 µL TE buffer.

Results

• Gel electrophoresis typically shows a high molecular weight DNA band with minimal smearing.

• DNA appears intact and suitable for PCR amplification.

Troubleshooting

Conclusion

The CTAB method is the benchmark for extracting high-quality DNA from plants. By meticulously adhering to the procedures and implementing the troubleshooting strategies, students are able to obtain DNA appropriate for molecular biology experiments, even from plants abundant in polysaccharides and polyphenols.

FAQs

Q1: Why is CTAB preferred for plant DNA extraction?
CTAB efficiently removes polysaccharides and polyphenols, common contaminants in plants, making it superior to simple detergent-based methods.

Q2: Can I use frozen plant tissue for DNA isolation?
Yes. In fact, freezing in liquid nitrogen helps preserve DNA integrity and makes grinding more effective.

Q3: How much tissue is needed per extraction?
Typically, 50–100 mg of fresh young leaves is sufficient to obtain good yields of DNA.

Q4: Why is β-mercaptoethanol added fresh?
It prevents oxidation of phenolic compounds, which can otherwise bind to DNA and degrade its quality.

Q5: What if DNA appears brown or degraded?
This often indicates polyphenol contamination. Increase PVP concentration or add antioxidants like ascorbic acid.

Q6: Can the extracted DNA be used for sequencing?
Yes, if purity is high (A260/280 ~1.8–2.0 and A260/230 ~2.0–2.4), the DNA is suitable for PCR, qPCR, and next-generation sequencing.

Q7: How do I confirm the quality of DNA?
Use spectrophotometric ratios and gel electrophoresis. High-molecular-weight intact DNA should appear as a clear band with minimal smearing.

Q8: How should DNA be stored after isolation?
DNA can be stored at 4 °C short-term (days to weeks) and −20 °C or −80 °C for long-term.

Q9: Can CTAB DNA be extracted from woody plants?
Yes, but buffer modifications like higher PVP (2–3%) and antioxidants may be required.

Q10: Is RNase treatment necessary?
Yes, RNA can interfere with DNA quantification and downstream applications, so RNase is recommended.