Experiment 5
(Lab Periods 5 and 6)
Gel Electrophoresis
A common method of analysis in molecular biology is Gel Electrophoresis. In
general, gel electrophoresis is a process by which the macromolecules within a sample
are separated from one another on the basis of size. The process can be applied to
different types of macromolecules such as proteins and nucleic acid (DNA and RNA). In
this lab you will use gel electrophoresis to determine whether or not the fragment of
DNA that you ‘amplified’ by PCR in experiment 4 is the expected size. Although this
will not constitute absolute proof, if the fragment present in your reaction tube is the
correct size it will strongly suggest that your PCR worked.
The basic idea behind gel electrophoresis is that you will force the macromolecules
through a porous matrix (the gel). The two most commonly used gels are composed of
either agarose - which you will use today - or acrylamide (polyacrylamide gels). DNA is
forced to move through the matrix by placing the gel in an electric field. DNA molecules
have a negative charge so they will flow from the negative pole to the positive pole of the
field. The DNA molecules, however, do not flow freely in the field. Instead, the gel acts
like a sieve to impede the migration of the DNA molecules, slowing the rate at which
they move towards the positive pole. The key to separating DNA molecules is that larger
molecules are more strongly impeded by the gel than smaller molecules. As a result,
smaller molecules of DNA (those composed of fewer nucleotides) migrate through the
gel at a faster rate. After a period of time, the shorter fragments are further from the
origin than are the longer molecules. In this manner, DNA fragments in a solution are
separated on the basis of size.
There are several basic steps to performing gel electrophoresis that will be
described below; 1) Pouring the gel, 2) Preparing your samples, 3) Loading the gel, 4)
Running the gel (exposing it to an electric field) and 5) Staining the gel.
1. To pour, or prepare, an agarose gel you boil a sample that is typically between
0.5% and 1% agarose (1% = 1g per 100ml). After it has boiled it is poured into
a mold and allowed to cool. After it has cooled it will solidify into a matrix.
When the gel is poured into the mold a comb is placed in the mold at one end so
that the gel contains several wells. This step will be completed before you come
to lab.
2. Preparing your sample means that you add a loading dye to your DNA
sample(s). The loading dye serves two purposes. The first is that it allows you to
visualize migration of the samples through the gel (the second function will be
discussed in step 3). Although you do not see the DNA molecules themselves
you can monitor the progress by observing how far the dye (which has a
negative charge) has migrated. The most typical dye is bromophenol blue which
migrates through a gel at the same rate as a DNA molecule that is 300
nucleotides in length.
