Fescue Chloroplast Genome Project - Part IV
Gel Electrophoresis
Goal – size separate DNA products in a gel matrix.
Materials
 A horizontal agarose gel
 Running buffer
 Your restriction digest from last week.
 Loading Dye
 Size Standard
 Ethidium Bromide (CAUTION – ethidum bromide is a mutagen, anyone handling
the gel or the running buffer must wear gloves)
Introduction
The restriction digest you set up last week should have cut your plasmid in at least two
places. In order to resolve and visualize those pieces we will separate them based on
their sizes in an agarose gel matrix.
Gel electrophoresis takes advantage of two phenomena,
1 – DNA has a net negative charge.
2 – Solidified agarose has pores large enough for DNA to travel through when
forced but not large enough for DNA to diffuse out.
The gel is a collection of carbohydrate strings that form an opaque jelly-like substance
when dissolved in water. Within the gel are randomly formed molecule sized spaces of
varying sizes. The overall negative charge of DNA means that in an electric field it will
move toward the positive pole. The electric field forces the DNA to negotiate its way
through those spaces. Small DNA pieces will move at a greater velocity than large ones.
If you care for an analogy…
Imagine trying to move through a thicket. Small mammals will travel through the
underbrush much more quickly than you will because they can find and move
through the more numerous small spaces between sticks and underbrush while
you’re stuck looking for much larger spaces.
Protocol
1. Add 2 μl of loading dye to your digest.
Loading dye = glycerol – increases the density of the sample so it sinks into the
well and then remains there
Bromophenol blue – dark blue dye, migrates at the same velocity
as a 300bp piece of DNA
Xylene cyanol – lighter blue than bromophenol, migrates at the
same velocity as a 4 kb piece of DNA
2. Pipette 15 μl of your digest/dye mixture into one well of the agarose gel prepared for
your class. The well farthest to the left will be filled with a mixture of size standards
(pieces of DNA of known lengths that will migrate in a predictable pattern and allow us
to estimate the sizes of our unknown pieces).
My sample is in lane ________
3. Turn on the power supply and run the gel during the lab period.
- electrode
wells
filled
with
DNA
100 volts
The DNA will be
invisible while the
gel is running so
dyes are included
in the loading
buffer to track the
progress of our
run.
Xylene cyanol
gel
When the power supply is
turned on an electric field
will be generated across the
gel and the DNA will move
out of the wells and towards
the positive electrode.
Bromophenol 
+ electrode
4. Since DNA is not visible, a special fluorescent stain (ethidium bromide) was added to
the gel. It fits specifically between stacked nucleotide bases and fluoresces orange when
illuminated with ultraviolet light.
Insert
Cloning vector
In visible light only
the tracking dyes are
visible.
In ultraviolet light DNA
will appear as orange
bands in the gel.
The cloning vector should appear as a band ~3 kb in every sample. Other bands are the
unknown insert. If an insert is present in the gel then your plasmid can be sequenced.
Cloning Part IV
Gel Electrophoresis
Lab Report
1. In an electric field, which pole will DNA migrate towards?
2. A pGemT plasmid with a putative insert was digested with EcoRI and the pieces
separated to give the result below. What is the size of the DNA insert in this plasmid?
3. Tape a picture of the gel run in your lab in the space below.
Which lane contains your plasmid?
Does your plasmid have an insert?
If so, estimate the size of the insert.