MEDI2011

• Agarose gel electrophoresis


  Most common form of electrophoresis run in a molecular lab
• Agarose


  • A polysaccharide isolated from seaweed
  • Supercoiled fibres form a meshwork of pores when solidified
  • Smaller molecules move through the pores more easily than larger molecules
• Agarose gel electrophoresis


  1. Negatively charged NAs placed on gel

  2. Electrical charge applied

  3. NAs migrate toward positively charged anode

  4. Degree of migration depends on size of NA

  
• Pore size


  • Dictated by agarose concentration
  • Higher [agarose] = smaller pores
• Agarose concentrations


  • 0.7-1.0% - NA ~800 bp-8kb
  • 1.2-1.5% - NA ~300 bp-5kb
  • 2% - NA ~0.1-2kb
• Electrophoresis buffers


  • TAE - 40 mM Tris-acetate, 1 mM EDTA
  • TBE - 45 mM Tris-borate, 1 mM EDTA
• Running a gel


  1. Dissolve agarose in electrophoresis buffer

  2. Heat mixture to dissolve agarose

  3. Cast gel in horizontal submarine system

  4. Run at 3-5 V/cm between electrodes
• Nucleic acid stains


  • Ethidium Bromide (EtBr)
  • SYBR Green/Gold/Safe
• Staining


  • Stain can be added to gel during casting or gel can be post-stained
  • Stains intercalate into major groove of NA

  
• Interpreting a gel


  1. Know expected product size

  2. Know size of each band in ladder/marker

  3. Run gel until ladder bands are resolved

  4. Identify ladder bands from top down

  5. Determine if correct product is present
• RNA agarose gel analysis

  • RNA can form secondary structures affecting migration
  • Formamide and formaldehyde added to denature RNA and maintain denatured state
• Polyacrylamide gel electrophoresis (PAGE)

  • Acrylamide and bisacrylamide polymerised to form cross-linked meshwork
  • Higher [acrylamide] = smaller pore size
  • Provides better resolution of small NA fragments
  • Not useful for large fragments
  • Gels must be post-stained

  
• Capillary electrophoresis


  • Variation of PAGE
  • Uses thin silicon tubes containing linear acrylamide polymer
  • Samples injected and electrophoresed under denaturing conditions
  • Smaller fragments travel faster than larger
  • Detector near anode detects DNA as it passes through
  • High voltage (100V+) can be applied
  • Fast, high resolution