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Background
Gel electrophoresis is an important molecular biology tool. Gel
electrophoresis enables us to study DNA. It can be used to determine
the sequence of nitrogen bases, the size of an insertion or deletion,
or the presence of a point mutation; it can also be used to distinguish
between variable sized alleles at a single locus and to assess
the quality and quantity of DNA present in a sample.
Gel electrophoresis is a method of separating chemical compounds
and molecules by their size and charge. The substances being separated
are placed in wells in an agarose gel and subjected to an electrical
field. Negatively charged molecules move towards the positive
anode, and positively charged molecules move towards the negative
anode. Longer or larger molecules have difficulty traveling through
the gel; they become entangled in the gel matrix. Shorter or smaller
molecules migrate through the agarose matrix faster and thus travel
farther in a given time period. Similar sized fragments travel
at relatively the same speed and form a tight "band"
when stained.
In these activities, we will introduce the concept of gel electrophoresis
and use it to determine the quantity and quality of the DNA the
students extracted. Electrophoresis also can be used in DNA sequencing,
profiling or "fingerprinting", and genetic engineering.
Overview of Activities
The activities are divided into three parts followed
by a student lab sheet:
Each portion can be accomplished in a 50-55 minute time period.
If time is short, you can prepare the gels (Activity 1) and stain
the gels (Activity 3) for the students.
Preparing an Agarose Gel
Different types and concentrations of media can be used to make
a gel. The concentration and type of media will affect the gel's
pore size and ability to separate similarly sized fragments. Agarose
gels separate DNA fragments differing by a hundred or more base
pairs, while polyacrylamide gels can separate DNA fragments differing
by a single base pair. Well forming combs are inserted into the
gel media as it cools and solidifies. After the gel solidifies,
the combs are removed. The gel will be placed into an electrophoresis
chamber, covered with a buffer solution, and DNA samples will
be loaded directly into the wells.
Preparing an agarose gel involves melting a specified amount of
agarose in TBE buffer, cooling the solution, and pouring it into
the gel casting tray. Gels solidify in 15-20 minutes. They can
be prepared by you ahead of time or students may prepare their
own (see Preparing an Agarose Gel).
Gels made ahead of time (1-2 days maximum) should be wrapped in
plastic wrap and stored in the refrigerator. Students do a great
job preparing gels, but sometimes it is easier and quicker to
prepare them yourself, especially if you have a limited number
of balances, microwaves, etc., or large classes. Advanced students
or student aides can also be assigned this task.
Loading an Agarose Gel
Gels are covered with a buffer solution. Prior to loading the
samples, the DNA must be mixed with a loading dye. The loading
dye serves two purposes: first, it increases the density of the
DNA so it will sink into the wells, and second, it provides a
visual marker so you know how far the DNA (which is not visible)
has traveled in the gel. Concentration and size standards are
loaded for comparison. Each DNA sample should be loaded into a
different lane (see Loading and Running an Agarose Gel).
Students have a tendency to stick the micropipettor into the well
while loading their samples. This causes two problems: first,
they can poke a hole in the bottom of the gel (their sample will
drain out the bottom into the buffer tank), and second, they create
so much turbulence within the well that the DNA sample squirts
out the top of the well. Remind them to keep the pipettor above
the well, but not in the well.
You may want to let students practice using the pipettors and
loading small volumes into the wells. Make practice gels using
agar (23g of agar per liter of distilled water) not agar.
Agar is much less expensive but cannot be used for running gels.
The agar should be melted, cooled to 55 C and then poured into
petri dishes. Suspend the combs in the petri dishes. Flood the
practice dishes with water. Students can practice loading colored
dyes mixed with glycerol or karo syrup. Common laboratory dyes,
such as crystal violet, methylene blue, safarin orange, methyl
orange, etc. make great practice dyes. Mix small amounts (1-2
mL) with an equal volume of karo syrup or glycerin.
Running an Agarose Gel
The buffer solution contains ions which conduct electrical charges.
The charges travel through the gel because it also contains ions
(it was prepared with the same buffer). DNA is negatively charged
and migrates toward the positive anode. The speed with which the
DNA moves through the gel is determined by the size of the DNA
fragment and the voltage. DNA will migrate faster at higher voltages
although lower voltages provide better resolution between similar
sized fragments. (The bands are less blurry.)
Students should run their gels for about 45 minutes or until the dye travels 1/2 - to - 3/4 of the way down the gel. They will have enough time to load and run their gels in one class period if they have had time to practice setting up the gel electrophoresis equipment, loading samples into agar petri dishes, know how to prepare their DNA and the agarose gels are ready to use. Otherwise, they can continue to run their gels after the class is over if you remember to check the gels and turn them off after about an hour (total running time). Students can tell a gel is running by looking for bubbles coming from the electrodes (once the power is on). They should never touch the gel boxes while the power is on because they can electrocute themselves if the top of the tank is open. Most buffer tanks have locking tops that prevent this.
Students should also load standards in order to determine the
quantity of DNA they extracted. These are DNA samples of known
concentrations that can be used for comparison. Typical standards
include: 50 ug/uL, 100 ug/uL and a 1 Kb ladder. The band produced
by the 100 ug/uL sample will be twice as dark as the band produced
by the 50 ug/uL sample. A 1 KB ladder produces numerous bands
of several different sizes. Concentration standards and ladders
can be ordered from molecular biology companies.
Staining the DNA in an Agarose Gel
DNA must be stained to be visible. Scientists use ethidium bromide
because it is highly sensitive to DNA and is visible under ultraviolet
light. Ethidium bromide is a mutagen and a carcinogen so it should
only be used with extreme care. Methylene blue, a dye, may also
be used to stain DNA. It is not harmful but requires the presence
of higher quantities of DNA to be visible (see Staining the DNA in an Agarose Gel).
Methylene blue will stain clothes, hands, and equipment. Spills
should be wiped up immediately and flushed with water.
The gels stain the best if they are in plastic container slightly
bigger than the gel itself. This also conserves the amount of
stain you use. Flood the gel with the stain. Gently rock the container
every 10 minutes to move the dye across the gel. Students can
do this section at the same time they are doing something else.
Study the gel and interpret the results:
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Science Education Connection Department of Biochemistry The University of Arizona Tuesday, January 14, 1997 warder@u.arizona.edu
http://biology.arizona.edu/sciconn/lessons/vuturo/ |
