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Agarose Gel Electrophoresis Essentials Guide

Learn the basics of agarose gel electrophoresis, including equipment, reagents, buffer systems, and troubleshooting tips. Understand how to separate DNA or RNA based on size, charge, or shape. Get insights on visualization, staining, and factors influencing DNA migration on agarose gels. Enhance your knowledge of molecular diagnostics and genomic DNA analysis.

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Agarose Gel Electrophoresis Essentials Guide

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  1. AGAROSE GEL ELECTROPHORESIS

  2. Terms • Gel electrophoresis is a method that uses an electrical current and a gel matrix to separate molecules like DNA and proteins. • Buffer a solution containing either a weak acid and its salt or a weak base and its salt, which is resistant to changes in pH.

  3. Agarose Gel Electrophoresis This is a procedure that separates molecules on the basis of their rate of movement through agarose gel under the influence of an electrical field. Separates DNA or RNA by: size and/or charge and/or shape

  4. Reagents and Supplies • Weighing scale • Spatula • Flask • Graduated cylinder • Microwave • Agarose • Buffer • Gel tray(s) and comb(s) • Gel box(es) • Power supply • DNA Staining solution • Photo doc. system

  5. Electrophoresis Equipment Power supply Cover Gel tank Electrical leads  Casting tray Gel combs

  6. Agarose • Agarose is a linear polymer extracted from seaweed. • An agarose gel is used to slow the movement of DNA . • Within an agarose gel, linear DNA migrate inversely proportional to their molecular weight.

  7. Resolution of linear DNA fragments in agarose gel % Agarose (w/v)Size Range (kb) for Optimal Separation 0.5 2 - 30 0.75 0.7 - 20 1.0 0.5 - 10 1.5 0.2 - 3 2.0 0.1 - 2

  8. Buffer Systems • Weak acids and/or bases that do not dissociate completely. • Purposes of buffer: • Maintain pH. • Generate ions consistently to maintain current & keep resistance low. • TAE, pH 8.0, ~50 mM - Tris, Acetate, EDTA • TBE, pH 8.0, ~50 mM - Tris, Borate, EDTA • TBE resolves low MW fragments better than TAE. • TAE resolves high MW fragments better than TBE • Tris (T) is a weak base. • Acetic (A) acid & boric (B) acid are weak acids.

  9. Pouring a horizontal agarose gel

  10. Visualization Monitoring the progress of the electrophoresis • Tracking dyes are visible to naked eye during run • Xylene cyanol (migrates with ~5.0 kb fragments) • Bromophenol blue (migrates with 300 bp fragments) • Orange G (migrates with fragments of ~50 bp) But • Mobility of tracking dyes can vary substantially depending on agarose • Concentration • Type

  11. Ethidium bromide staining • Binds to ds DNA by intercalation between stacked bases. • Used to visualize DNA with UV light. ***CAUTION! • UV light damages eyes and skin! Wear goggles and/or face shield. • Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn at all times.

  12. DNA - + Power How fast will the DNA migrate? • Strength of the electrical field • Size of the DNA • Buffer • Concentration of agarose gel used small large

  13. What factors affect mobility of linear ds DNA? • Pore size of the gel •  [agarose]   pore size •  pore size   friction   mobility • Voltage across the gel •  voltage   mobility • Length of the DNA molecule • smaller molecules generate less friction and so move faster

  14. Factors affecting resolution Resolution = separation of fragments The “higher” the resolution, the more space between fragments of similar, but different, lengths. Resolution is affected by • agarose type & concentration • salt concentration of buffer or sample • amount of DNA loaded in the sample • voltage

  15. Why run an agarose gel? • Determine the quality or quantity of DNA • Estimate the size of DNA molecules • Purification of DNA • Analyze PCR products • Molecular diagnosis or genotyping

  16. Genomic DNA M = 1kb + DNA ruler

  17. PCR products M 3a 4a 4b 1a 1b 2a

  18. Msel digestion of PCR products

  19. PCR products

  20. PCR products

  21. Genomic DNA

  22. Trouble shooting • Smearing • torn sample wells • voltage too high for large fragments • too much DNA • Gel melts • voltage too high • ionic strength too low • Poor resolution • wrong agarose concentration • small bands are fuzzy –diffusion of the DNA and broadening of the band

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