University of Pittsburgh at Bradford
Science In Motion
Biology Lab 004
Introductory Agarose Gel Electrophoresis
Introduction:
Agarose gel electrophoresis is a widely used method that separates molecules based upon charge,
size and shape. It is particularly useful in separating charged biomolecules such as DNA, RNA and
proteins.
Agarose gel electrophoresis possesses great resolving power, yet is relatively simple and
straightforward to perform. The gel is made by dissolving agarose powder in boiling buffer solution. The
solution is then cooled to approximately 55oC and poured into a mold where it solidifies. The gel is
submerged in a buffer-filled chamber, which contains electrodes.
Samples are prepared for electrophoresis by mixing them with components that will give the
mixture density, such as glycerol or sucrose. This makes the sample denser than the electrophoresis
buffer. These samples can then be loaded with a micropipet or transfer pipet into wells that were created
in the gel by a template during casting. The dense samples sink through the buffer and remain in the
wells.
A direct current power supply is connected to the electrophoresis apparatus and current is applied.
Charged molecules in the sample enter the gel through the walls of the wells. Molecules having a net
negative charge migrate towards the positive electrode (anode) while net positively charged molecules
migrate towards the negative electrode (cathode). Within a range, the higher the applied voltage, the
faster the samples migrate. The buffer serves as a conductor of electricity and to control the pH. The pH
is important to the charge and stability of biological molecules.
Agarose is a polysaccharide derivative of agar. It contains microscopic pores, which act as a
molecular sieve. The sieving properties of the gel influence the rate at which a molecule migrates.
Smaller molecules move through the pores more easily than larger ones. Molecules can have the same
molecular weight and charge but different shapes. Molecules having a more compact shape (a sphere is
more compact than a rod) can move more easily through the pores.
Factors such as charge, size and shape, together with buffer conditions, gel concentrations and
voltage, affect the mobility of molecules in gels. Given two molecules of the same molecular weight and
shape, the one with the greater amount of charge will migrate faster. In addition, different molecules can
interact with agarose to varying degrees. Molecules that bind more strongly to the agarose will migrate
more slowly.
Objective:
To develop a basic understanding of electrophoretic theory, and to gain “hands-on” familiarity
with the procedures involved in agarose gel electrophoresis to separate different molecules.
Safety:
Gloves and safety goggles should be worn routinely as good laboratory practice.
Materials:
electrophoresis apparatus (includes gel casting tray, dams, and comb)
250 ml flask, graduated cylinder, pipet, buffer concentrate, agar, distilled water
dye samples A-F
power source, analytical balance, microwave
Adapted from Edvotek: Introduction to Electrophoresis
1
Procedure:
Part 1 (this may already be done for you)
1. Preparing the gel bed:
a. Close off the open ends of a clean and dry casting tray by using rubber dams or tape.
i. Rubber dam: place a rubber dam on each end of the bed. Make sure the dam sits
firmly in contact with the sides and bottom of bed.
ii. Taping: with ¾ inch wide tape, extend the tape over the sides and bottom edge of bed.
Fold the extended edges of the tape back onto the sides and bottom. Press contact
points firmly to form a good seal.
b. Place a well-former template (comb) in the set of notches. Make sure the comb sits firmly and
evenly across the bed.
2. Casting the gel: (this experiment requires a 0.8% gel)
a. Use a 250ml flask to prepare the diluted gel buffer.
i. With a micropipet, measure 600 l of the buffer concentrate and add 29.4 ml of
distilled water to the beaker.
ii. Add 0.24 grams of agarose powder. Swirl to disperse the clumps.
iii. With a marking pen, indicate the level of the solution volume on the outside of the
flask.
b. Heat the mixture to dissolve the agarose powder. The final solution should be clear (like
water) without any undissolved particles.
i. Cover flask with plastic wrap to prevent evaporation.
ii. Heat the mixture on high for 1 minute
iii. Swirl the mixture and heat on high in bursts of 25 seconds until all the agarose is
completely dissolved.
c. Cool the agarose solution to 55oC with careful swirling to promote even dissipation of heat. If
detectable evaporation has occurred, add distilled water to bring the solution up to the original
volume.
d. Pour the cooled agarose solution into the bed. Make sure the bed is on a level surface.
e. Allow the gel to completely solidify. It will become firm and cool to the touch after
approximately 20 minutes.
Part 2
3. Preparing the gel for electrophoresis:
a. Carefully and slowly remove the rubber dams or tape.
b. Remove the comb by slowly pulling it straight up. Do this carefully and evenly to prevent
tearing the sample wells.
c. Place the gel with the gel tray in the electrophoresis chamber, properly oriented, centered and
level on the platform (most molecules will run from negative to positive so the wells should be
located by the negative electrode).
Adapted from Edvotek: Introduction to Electrophoresis
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d. Fill the chamber of the electrophoresis apparatus with buffer solution so that it covers the gel.
e. Load samples in wells and conduct electrophoresis as follows:
4. Conducting agarose gel electrophoresis:
a. Load each of the samples in tubes A-F, using the automatic micropipets, into the wells in
consecutive order. The amount of sample that should be loaded is 15l.
b. After the samples are loaded, carefully snap the cover down onto the electrode terminals.
Make sure the negative and positive indicators on the cover and apparatus chamber are
properly oriented.
c. Insert the plug of the black wire into the black input of the power source (negative input).
Insert the plug of the red wire into the red input of the power source (positive input).
d. Set the power source at 125 volts and run the electrophoresis for 30-45 minutes.
e. Check to see that the current is flowing properly – you should see bubbles forming on the
electrodes.
f. After electrophoresis is completed, turn off the power, unplug the power source, disconnect the
leads and remove the cover.
g. Examine your results and answer the following questions.
Adapted from Edvotek: Introduction to Electrophoresis
3
Name________________________
Introductory Agarose Gel Electrophoresis
Student Evaluation
Questions:
1. On what basis does agarose gel electrophoresis separate molecules?
2. Explain migration according to charge.
3. What conclusions can be drawn from the results of sample F?
4. Why is glycerol added to the solutions before they are loaded into the wells?
5. What would happen if distilled water were substituted fro buffer in either the chamber solution or the
gel solution?
Adapted from Edvotek: Introduction to Electrophoresis
4