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.

Agarose gel electrophoresis is a method to separate DNA, or RNA molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). Shorter molecules move faster and migrate farther than longer ones . Separates DNA or RNA by: size and/or charge and/or shape

Electrophoresis Equipment Reagents and Supplies Power supply 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 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 . 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 2 - 30 0.75 0.7 - 20 1.0 0.5 - 10 1.5 0.2 - 3 2.0 0.1 - 2

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.

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, GelRed ***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. Although the stained nucleic acid fluoresces reddish-orange, images are usually shown in black and white.

Visualisation: Ethidium Bromide (EtBr) and dyes The most common dye used to make DNA or RNA bands visible for agarose gel electrophoresis is ethidium bromide, usually abbreviated as EtBr. It fluoresces under UV light when intercalated into DNA (or RNA). By running DNA through an EtBr-treated gel and visualizing it with UV light. EtBr is a known mutagen, however, safer alternatives are available. Buffers The most common being: Tris/Acetate/EDTA (TAE)

Procedure Make a 1% agarose solution in 20ml TAE. （0.2g agarose in 20ml TAE ） Carefully bring the solution just to the boil to dissolve the agarose. Let the solution cool down to about 60 °C at room temperature. Stir or swirl the solution while cooling. Add ethidium bromide stock in the gel solution for a final concentration of 0.5 ug/ml. Be very careful when handling the concentrated stock. Stir the solution to disperse the ethidium bromide, then pour it into the gel rack. Insert the comb at one side of the gel, about 5-10 mm from the end of the gel. When the gel has cooled down and become solid, carefully remove the comb. The holes that remain in the gel are the wells or slots. Put the gel, together with the rack, into a tank with TAE. The gel must be completely covered with TAE, with the slots at the end electrode that will have the negative current.

+ - How fast will the DNA migrate? Strength of the electrical field Size of the DNA Buffer Concentration of agarose gel used DNA + - Power small 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 agarose concentration salt concentration of buffer or sample amount of loaded DNA voltage

Sample Preparation Mix the samples of DNA with the 6X sample loading buffer (w/ tracking dye). This allows the samples to be seen when loading onto the gel, and increases the density of the samples, causing them to sink into the gel wells. 6X Loading Buffer:   Bromophenol Blue (for color)  Glycerol (for weight)

Loading the Gel Carefully place the pipette tip over a well and gently expel the sample. The sample should sink into the well. Be careful not to puncture the gel with the pipette tip.

Running the Gel Place the cover on the electrophoresis chamber, connecting the electrical leads. Connect the electrical leads to the power supply. Be sure the leads are attached correctly - DNA migrates toward the anode (red). When the power is turned on, bubbles should form on the electrodes in the electrophoresis chamber.

Cathode (-)  wells DNA (-)   Bromophenol Blue Gel Anode (+) After the current is applied, make sure the Gel is running in the correct direction. Bromophenol blue will run in the same direction as the DNA.

DNA Ladder Standard DNA migration -  12,000 bp  5,000  2,000  100  200  300  1,650  1,000  500  850  650  400  12,000 bp  5,000  2,000 DNA migration Note: bromophenol blue migrates at approximately the same rate as a 300 bp DNA molecule bromophenol blue + Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy to determine the sizes of unknown DNAs.

As an alternative to purchasing costly DNA ladders, one can be created using meal worm (Tenebrio molitor) DNA and a restriction enzyme. http://people.uis.edu/rmosh1/DNAexerciseVIIa02.pdf