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Gel Electrophoresis: Separating DNA Fragments by Size

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Gel electrophoresis is a technique that separates DNA fragments by size using an electric field. DNA pieces move through a jelly-like gel, and smaller pieces travel farther than larger ones in the same time.

This matters because after cutting or copying DNA, scientists must check what sizes are present. The separated bands act like a barcode that shows whether a gene insert is correct, whether PCR worked, or whether samples differ.

Key Concepts

Agarose gel is a porous gel made from seaweed extract, used for DNA. Wells are slots where samples are loaded. DNA is negatively charged, so it moves toward the positive electrode. Buffer carries current and keeps pH steady. DNA ladder is a standard mix of known sizes for comparison. Ethidium bromide or safer dyes make bands visible under ultraviolet or blue light. Sizes are measured in base pairs (bp).

How It Works

  1. Make agarose gel with wells and place it in buffer.
  2. Mix DNA samples with loading dye and put them into wells.
  3. Also load a DNA ladder of known fragment sizes.
  4. Apply voltage; DNA moves toward the positive end.
  5. Run until dye has moved a suitable distance, then stop.
  6. Stain and view bands; estimate sizes against the ladder.

Worked Example

A ladder has bands at 100, 200, 300, 500 and 1,000 base pairs. An unknown PCR band travels the same distance as halfway between the 300 and 500 base pair bands on a gel where distance roughly follows log of size. Step 1: log10 of 300 is about 2.48 and log10 of 500 is about 2.70. Step 2: midpoint = (2.48 + 2.70) / 2 = 2.59. Step 3: 10 to the power 2.59 is about 389. So the unknown fragment is roughly 390 base pairs, close to the expected 400 base pair product.

Common Misconceptions

  • Larger DNA moves faster. Larger fragments are held back by the gel and move more slowly.
  • DNA moves to the negative electrode. DNA is negatively charged, so it moves to the positive electrode.
  • One band always means one fragment size only. Two fragments of very similar size can overlap in a single band.

Real-World Applications

Gel electrophoresis checks PCR products, verifies restriction digests and plasmid inserts, separates DNA for fingerprinting, tests seed and food samples for specific DNA, and is used in forensic and disease-testing laboratories every day.

In summary, gel electrophoresis sorts DNA by size through a gel under electric current. Comparing bands with a ladder turns invisible DNA pieces into readable results.

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