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
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 ? 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 ml 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).
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 15ml.
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.
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