DNA Restriction Digestion and Agarose Gel Electrophoresis

Posted on Mar 17, 2026 in Biotechnology

Understanding Restriction Enzymes

Restriction enzymes (RE), or restriction endonucleases, act as molecular scissors by cutting DNA at specific recognition sequences. These enzymes are isolated from various cellular strains and typically recognize palindromic sequences of 4 to 6 base pairs. Once the target site is located, the enzyme cleaves both strands of the DNA double helix, resulting in restriction fragments.

Common Restriction Enzymes

Every enzyme has unique target sites. For example, Lambda DNA contains multiple sites for:

  • EcoRI: Recognizes 5′-GAATTC-3′
  • HindIII: Recognizes 5′-AAGCTT-3′

After digestion, fragments are separated by agarose gel electrophoresis, where smaller fragments migrate faster than larger ones. Fragment sizes are determined by comparing them against a DNA molecular weight marker (ladder).

Materials Required

Chemicals and Reagents

  • Lambda (λ) DNA
  • Restriction enzymes: EcoRI and HindIII
  • 10X Assay buffers (EcoRI and HindIII)
  • Molecular biology grade water
  • Agarose and 50X TAE buffer
  • 6X Gel loading buffer
  • DNA marker (ladder)
  • Ethidium bromide

Equipment

  • Micropipettes and tips
  • Microcentrifuge tubes
  • Water bath/incubator (37°C)
  • Electrophoresis unit and power supply
  • UV transilluminator
  • Gel casting tray and comb

Procedure: Restriction Digestion

Reaction Setup:

  • Lambda DNA: 5 µl
  • 10X Buffer: 2.5 µl
  • Molecular biology grade water: 16.5 µl
  • Restriction Enzyme: 1 µl
  • Total Volume: 25 µl
  1. Mix reaction components gently by tapping and brief centrifugation.
  2. Incubate at 37°C for 1 hour in a water bath.
  3. Keep tubes at room temperature for 10 minutes post-incubation.

Agarose Gel Electrophoresis

Principle

Agarose gels act as molecular sieves. Because DNA is negatively charged due to its phosphate backbone, it migrates toward the positive electrode (anode). Separation is determined by charge, size, and shape.

Gel Preparation and Loading

  1. Prepare 1% agarose gel by dissolving 0.5 g agarose in 50 ml of 1X TAE buffer.
  2. Heat until dissolved, cool to 55–60°C, and add ethidium bromide.
  3. Pour into a casting tray with a comb and allow to solidify.
  4. Mix 10 µl of digested sample with 2 µl of 6X gel loading dye.
  5. Load 3 µl of DNA marker into the first well, followed by samples.
  6. Run electrophoresis at 80–100 V.
  7. Visualize bands under a UV transilluminator.

Precautions

  • Keep enzymes and buffers on ice.
  • Add enzymes last to the reaction mixture.
  • Use fresh tips for each reagent to avoid contamination.
  • Do not interchange buffers between enzymes.
  • Handle ethidium bromide with extreme care (mutagenic/carcinogenic).
  • Do not exceed incubation times to prevent star activity.