Agarose Gel Electrophoresis
Electrophoresis is the movement of charged particles in solution under the influence of an
electric field. In the most common form of electrophoresis, the sample is applied to a
stabilizing medium which serves as a matrix for the buffer in which the sample molecules
will travel. The agarose gel is a common type of stabilizing medium used for the
electrophoretic separation of nucleic acids. A diagram of the essential components of an
agarose electrophoretic system is shown below. The agarose gel, containing preformed
sample wells, is submerged in buffer within the electrophoretic gel cell. Samples to be
separated are then loaded into the sample wells. Current from the power supply travels to
the negative electrode (cathode), supplying electrons to the conductive buffer solution,
gel and positive electrode (anode), thus completing the circuit.
At neutral pH, a molecule of DNA or RNA is negatively charged because of the
negative charges on the phosphate backbone. Under these conditions, nucleic acids
applied to sample wells at the negative electrode end of the gel migrate within pores of
the gel matrix towards the positive electrode. The agarose gel serves as a molecular sieve
in that its structure is similar to that of a sponge. Agarose is the neutral fraction of agar,
made up of linear molecules consisting of repeating units of the disaccharide agarbiose.
Agarose solutions undergo a solution-gel transition at 45ºC or below. This gelling
property, as well as the high gel strength obtained at low concentrations, makes the
agarose gel a most useful separation medium.
The primary mechanism for the formation of agarose gels based on X-ray analysis
has been postulated as the formation of double helices. Secondary and tertiary
contributions arise from hydrogen bonding and hydrophobic interactions, respectively.
The result is a thermally reversible gel which is slightly opaque due to the molecular
aggregates or “microcrystallites” which hold the gel together.
Resolving power
The resolving power of an agarose gel depends on the pore size, which is dictated
by the concentration of dissolved agarose. Generally, large molecules move more slowly
through the gel than smaller molecules. Thus, the method sorts the molecules according
to size, since it relies on the ability of uniformly charged nucleic acids to fit through the
pores of the agarose gel matrix. High percentage agarose gels (e.g. 1.5%) are used for the
separation of small DNA molecules (100 – 1000 base-pairs in length), while low
percentage gels (e.g. 0.6%) are used for large molecules (104 – 105 base-pairs).
The following is a table that will help you decide what concentration your gel should be.
How to Pour an Agarose Gel
In this lab, agarose gels will always be made in 0.04M Tris-Acetate-EDTA, pH 8.3 (1X
TAE) buffer. The percentage gel (w/v) will very depending on the size of molecules we
are trying to resolve.
1. Weigh out the appropriate amount of agarose (ex. a 1.5% gel would be 1.5g agarose in
100 mL). Usually we will make 40-50 mL of gel solution. Add the appropriate amount
of 1X TAE. Make the mixture in a 250 mL flask, cover it with Saran Wrap, and
microwave for 1 minute and 20 seconds on high power.
2. Visually check to see that all the agarose has melted. Unmelted agarose looks like
tiny refractive lenses floating around. If not completely melted, nuke it a little longer.
Try 20 second intervals.
3. Allow the gel to cool a bit (5 minutes on the bench is usually sufficient). If called for,
add 0.5 μL of 10 mg/mL ethidium bromide, and then pour the gel into the mold. Pour up
to half the height of the teeth on the comb. Chase out any bubbles on the surface with a
plastic Pasteur pipet or micropipettor tip.
4. Allow the gel to cool until it turns slightly white. This usually takes at least 20
minutes at room temperature. Remove the comb, and pour enough 1X TAE into the
buffer chamber to barely cover the top surface of the gel. The gel may now be loaded
with your samples.