Agarose gel electrophoresis

Principle

• Nucleic acids carry a negative charge from their phosphate backbone and migrate toward the anode (positive electrode) in an electric field.
• The agarose matrix acts as a sieve: smaller fragments move faster; migration is roughly inversely proportional to the log of fragment size.
• Typical agarose concentration 0.7–2%: lower percentages resolve large fragments, higher percentages resolve small PCR products.

Reagents and equipment

• Agarose, running buffer (TAE or TBE), gel tray, comb, tank and power supply.
• Loading dye with glycerol or Ficoll (makes sample sink) and tracking dyes (bromophenol blue, xylene cyanol).
• DNA ladder (molecular weight marker).
• Stains: ethidium bromide (intercalator, orange fluorescence under UV; mutagenic), or safer dyes such as SYBR Safe or GelRed.

Procedure

• Dissolve agarose in buffer by heating, cool to about 55–60 °C, add stain if in-gel, pour with comb, allow to set.
• Submerge gel in buffer with wells at the cathode (negative, black) end; "run to red".
• Load ladder, samples and controls mixed with loading dye.
• Run at about 5–8 V/cm until dye front has migrated sufficiently.
• Visualise on UV or blue-light transilluminator and document.

Results

• Estimate fragment size by comparing with the ladder.
• Plasmid forms run differently: supercoiled usually fastest, then linear, then nicked (open circular).
• Intact total RNA shows sharp 28S and 18S rRNA bands in roughly 2:1 intensity; RNA is best run on denaturing gels.

Quality control and pitfalls

• Smearing: degraded DNA, overloading, too high voltage or salt.
• No bands: failed PCR, wrong polarity, missing stain.
• Primer dimers appear as diffuse bands below about 100 bp.
• Wear gloves and eye/UV protection; dispose of ethidium bromide as hazardous waste.

Clinical use

• Checking PCR products, DNA/RNA integrity, restriction fragment analysis (PCR-RFLP) and gel purification.