DNA isolation is a fundamental technique in molecular biology, essential for genetic analysis, marker studies, and molecular breeding. In eukaryotic cells, chromosomal DNA resides within the nucleus, protected by a cell wall and plasma membrane in plants. This guide describes an efficient DNA extraction protocol using the Cetyl Trimethyl Ammonium Bromide (CTAB) method, based on the procedure by Doyle and Doyle (1987), with modifications suited for green gram seedlings.


Principle of DNA Isolation

The DNA isolation process involves several key steps to ensure high-quality DNA yield:

  1. Cell wall disruption: Mechanical grinding breaks the cell wall, releasing the cellular contents.
  2. Cell membrane lysis: CTAB, a cationic detergent, binds to polysaccharides in high-salt conditions, aiding in their removal while releasing the DNA.
  3. Nuclease inhibition: EDTA chelates Mg²⁺ ions, which are essential cofactors for nucleases, preventing DNA degradation.
  4. Protein denaturation: A chloroform:isoamyl alcohol mixture denatures and separates proteins from nucleic acids.
  5. DNA precipitation: Isopropanol precipitates DNA, allowing it to be recovered in pellet form.


Materials and Reagents

Equipment Needed

  • Liquid nitrogen
  • Pestle and mortar
  • Water bath
  • Cheesecloth
  • Funnel
  • Centrifuge and centrifuge tubes
  • Beakers
  • Conical flask
  • Pipettes
  • Eppendorf tubes
  • Ice bath


Reagents Needed

1. Extraction Buffer:

  • 100 mM Tris
  • 20 mM EDTA
  • 1.4 M NaCl
  • 3% CTAB

2. Other Chemicals:

  • PVP (Polyvinylpyrrolidone)
  • β-mercaptoethanol
  • Chloroform: isoamyl alcohol (24:1)
  • Chilled isopropanol
  • T.E buffer (Tris-EDTA, 10:1)
  • RNase A (100 mg/ml)
  • Phenol
  • Absolute ethanol
  • 70% ethanol


Protocol for DNA Extraction (Green Gram)

1. Sample Preparation

  1. Collect 2 g of young, healthy leaves from 15 to 20-day-old green gram seedlings.
  2. Clean the leaves with distilled water and wipe with 70% ethanol.
  3. Remove the midrib and homogenize the leaf tissue in liquid nitrogen using a pestle and mortar to obtain a fine powder.


2. DNA Extraction Process

  1. Cell Lysis:

    • Transfer the powdered leaf tissue to a 50 ml Falcon tube containing 12 ml extraction buffer (20 mM EDTA, 1.4 M NaCl, 100 mM Tris, 3% CTAB).
    • Add a pinch of PVP to bind polyphenols and 50 µl of β-mercaptoethanol to break disulfide bonds in proteins.

  2. Incubation:

    • Incubate the sample at 65°C for 1 hour, ensuring occasional mixing for uniform lysis.

  3. Protein Removal:

    • Add 5 ml of chloroform:isoamyl alcohol (24:1) to the lysate and mix gently to emulsify the solution.
    • Centrifuge the sample at 8000 rpm for 15 minutes at 4°C to separate the aqueous phase (containing DNA) from the organic phase (proteins and debris).
    • Carefully transfer the aqueous phase to a fresh tube.

  4. DNA Precipitation:

    • Add 2.5 volumes of chilled isopropanol to the aqueous phase.
    • Incubate the mixture overnight at 4°C for effective DNA precipitation.

  5. Pellet Collection:

    • Centrifuge the mixture again at 8000 rpm for 15 minutes at 4°C to collect the DNA pellet.
    • Discard the supernatant and wash the pellet with 70% ethanol to remove salts.

  6. Drying the Pellet:

    • Air dry the pellet to remove residual ethanol without over-drying, which may make DNA difficult to dissolve.

  7. DNA Resuspension:

    • Dissolve the dried DNA pellet in 100-200 µl of T.E buffer (Tris-EDTA) depending on the pellet size.
    • For RNA-free DNA, treat the sample with RNase A (100 mg/ml) at 37°C for 30 minutes.


Conclusion

This CTAB-based DNA extraction method is reliable and effective for isolating high-quality DNA from green gram leaves. It ensures the removal of polysaccharides, polyphenols, and proteins — common inhibitors in plant DNA extraction — making it suitable for downstream molecular applications such as PCR, qPCR, and genomic library construction.

By following this streamlined protocol, researchers can ensure reproducible results, supporting accurate genetic analysis and plant breeding studies.