AGAROSE GEL ELECTROPHORESIS IMBB 2016 Bec AILRI Hub
AGAROSE GEL ELECTROPHORESIS IMBB 2016 Bec. A-ILRI Hub, Nairobi May 9 – 20, 2016 Martina Kyalo
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 p. H.
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: ü size and/or ü charge and/or ü shape
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
Electrophoresis Equipment Power supply Cover Gel tank Electrical leads Casting tray Gel combs
Agarose • Agarose is a linear polymer extracted from seaweed. • An agarose gel is used to slow the movement of DNA. ü Within an agarose gel, linear DNA migrate inversely proportional to their molecular weight.
Resolution of linear DNA fragments in agarose gel % Agarose (w/v) Size Range (kb) for Optimal Separation 0. 5 0. 75 1. 0 1. 5 2. 0 2 - 30 0. 7 - 20 0. 5 - 10 0. 2 - 3 0. 1 - 2
Buffer Systems • Weak acids and/or bases that do not dissociate completely. • Purposes of buffer: – Maintain p. H. – Generate ions consistently to maintain current & keep resistance low. 1) TAE, p. H 8. 0, ~50 m. M - Tris, Acetate, EDTA 2) TBE, p. H 8. 0, ~50 m. M - Tris, Borate, EDTA Ø Ø • • TBE resolves low MW fragments better than TAE resolves high MW fragments better than TBE Tris (T) is a weak base. Acetic (A) acid & boric (B) acid are weak acids.
Pouring a horizontal agarose gel
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
DNA stain • Binds to ds DNA by intercalation between stacked bases. • Used to visualize DNA with UV light. – E. g. Ethidium bromide, Gel. Red ***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.
How fast will the DNA migrate? ü Strength of the electrical field ü Size of the DNA ü Buffer ü Concentration of agarose gel used DNA small - Power large +
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
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 üagarose concentration üsalt concentration of buffer or sample üamount of loaded DNA üvoltage
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
Genomic DNA M 1 2 3 M = 1 kb + DNA ruler 1 = Lambda DNA (control) 2 - 3 = g. DNA
PCR products M M 3 a 4 a 4 b 1 a = 1 kb + DNA ruler 1 b 2 a
Msel digestion of PCR products M = 1 kb + DNA ruler
Genomic DNA 1 = Lambda DNA (control) 2 – 13 = g. DNA
PCR products M = 1 kb + DNA ruler
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
- Slides: 21
