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 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

4. 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

5. 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.

6. Resolution of linear DNA fragments in agarose gel
% Agarose (w/v) Size Range (kb)
for Optimal Separation

7. 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.

8. Pouring a horizontal agarose gel

9. 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

10. 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.

11. 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)

12. 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.

13. + - 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

14. 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

15. 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

16. 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)

17. 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.

18. 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.

19. 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.

20. 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.

21. As an alternative to purchasing costly DNA ladders, one can be created using meal worm (Tenebrio molitor) DNA and a restriction enzyme.