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AGAROSE GEL ELECTROPHORESIS

AGAROSE GEL ELECTROPHORESIS. 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.

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AGAROSE GEL ELECTROPHORESIS

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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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