Gel Electrophoresis Quick Notes

Big Ideas
  • Gel electrophoresis separates charged molecules using an electric field and a porous gel matrix.
  • Agarose gel electrophoresis is standard for analyzing large, linear DNA fragments.
  • Separation is based primarily on molecular size: shorter DNA moves farther toward the positive electrode.
Mechanism of Separation
  • DNA carries a uniform negative charge from its phosphate backbone.
  • When voltage is applied, DNA migrates toward the anode (positive pole).
  • The agarose network hinders movement; longer fragments experience more friction and lag behind shorter ones.
Agarose Gel Properties
  • Composed of seaweed-derived polysaccharide fibers suspended in buffered solution.
  • Pore size is adjustable by changing agarose concentration; lower concentration yields larger pores suitable for bigger DNA.
  • Gel acts like a molecular sieve, imposing size-dependent resistance on migrating DNA.
DNA Ladder (Molecular Ruler)
  • Mixture of DNA fragments with known lengths loaded beside samples.
  • Band positions create a reference scale to estimate sizes of unknown PCR products quickly.
Visualizing DNA
  • Intercalating dyes (e.g., ethidium bromide, Gel Red) insert between base pairs.
  • Upon ultraviolet illumination, bound dye fluoresces, revealing DNA bands.
  • Safety: handle dyes and UV sources with proper protection.
Loading Dye
  • Contains dense agents such as glycerol or sucrose to sink samples into wells.
  • Includes visible tracking dyes that migrate with the electrical front, indicating run progress.
Pipetting into Wells
  • Steady both hands to avoid puncturing gel bottom.
  • Insert tip just below buffer surface, above well base.
  • Depress plunger only after the tip is positioned; release after withdrawing to prevent suction.
Quick Recall Checklist
  • DNA → negative; moves to positive electrode.
  • Longer fragments = slower; shorter fragments = faster.
  • Agarose concentration tunes resolution range.
  • Always load ladder for size estimation.
  • Use intercalating dye plus UV to see bands.
  • Loading dye: weight + color; sink and track.
  • Gentle pipetting prevents well damage and sample loss.