Agarose Gel, Ethidium Bromide and UV Visualisation
Biology · Biotechnology: Principles and Processes · NEET
DNA fragments are separated on an agarose gel: because DNA is negatively charged, it moves toward the positive electrode (anode), and smaller fragments travel farther. The separated DNA cannot be seen in normal light, so it is stained with ethidium bromide and viewed under UV light, where it shows bright orange bands. Memory hook: "Agarose sieves, Ethidium stains, UV reveals orange."
DNA is negatively charged, so it moves from the cathode toward the anode through the agarose mesh; smaller fragments travel farther. Bands are stained with ethidium bromide and viewed under UV light as bright orange.
Your doubts, answered
Why is agarose (and not just any gel) used?
Agarose is a natural polymer taken from sea weeds. It forms a mesh-like matrix that acts as a molecular sieve. The concentration of agarose sets how tight the mesh is, so it controls how well fragments of different sizes separate. This is why NCERT names agarose specifically.
Why does DNA move, and in which direction?
DNA carries a negative charge because of its phosphate groups. When current is passed, DNA is pulled toward the positive electrode, called the anode. So DNA always moves away from the negative electrode (cathode) toward the anode.
Why do smaller fragments move farther?
The agarose mesh slows down movement. Small fragments slip through the mesh easily and travel a long distance. Large fragments get held up by the mesh and stay near the top. So distance moved is inversely related to size: smaller = farther.
Why can't we see the DNA bands directly?
Pure DNA is colourless and cannot be seen with the naked eye in the gel. That is why a staining step is needed. Without staining, you would not know where the separated fragments are sitting in the gel.
What exactly does ethidium bromide do?
Ethidium bromide is a dye (a stain) that binds to DNA. It is not a light. After running the gel, you soak the gel in ethidium bromide, and it sticks to the DNA fragments. Then, under UV light, only the DNA-bound dye glows.
Green or orange? What colour appears?
Under UV radiation, ethidium-bromide-stained DNA shows bright ORANGE coloured bands. NEET has directly asked this colour (2023). Do not write green, red, blue or yellow for the NCERT answer.
UV or infrared light?
The bands are seen under ULTRAVIOLET (UV) radiation, not infrared and not ordinary visible light. UV is the energy that makes the ethidium-bromide-DNA complex fluoresce.
⚠️ The NEET trap ✗ Ethidium bromide is a light source, and DNA glows green under infrared radiation. ✓ Ethidium bromide is a stain (dye) that binds DNA; the bands are seen as bright orange under UV radiation. 🧠 NTA loves to swap two things at once: the light (UV, not infrared) and the colour (orange, not green). Lock both: UV light plus orange bands.
Real NEET questions
NEET 2017
What is the criterion for DNA fragments movement on agarose gel during gel electrophoresis?
A · The larger the fragment size, the farther it moves
B · The smaller the fragment size, the farther it moves ✓
C · Positively charged fragments move to farther end
D · Negatively charged fragments do not move
Solution: Negatively charged DNA moves toward the anode through the agarose mesh, which acts as a sieve. Smaller fragments face less resistance and migrate farther, so the smaller the fragment, the farther it moves.
NEET 2020
In gel electrophoresis, separated DNA fragments can be visualized with the help of
A · Acetocarmine in UV radiation
B · Ethidium bromide in infrared radiation
C · Acetocarmine in bright blue light
D · Ethidium bromide in UV radiation ✓
Solution: DNA is stained with the dye ethidium bromide and then exposed to UV radiation to make it visible. Acetocarmine (a chromosome stain) and infrared/blue-light options are wrong.
NEET 2023 Phase 1
Upon exposure to UV radiation, DNA stained with ethidium bromide will show
A · Bright red colour
B · Bright blue colour
C · Bright yellow colour
D · Bright orange colour ✓
Solution: Ethidium-bromide-stained DNA fluoresces under UV light as bright orange coloured bands, exactly as stated in NCERT. Red, blue and yellow are distractors.
Solved Biotechnology: Principles and Processes NEET PYQs
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