Fragments of DNA can be separated based on size by gel electrophoresis. After the DNA is inserted into a well within the gel matrix, an electrical current is applied to the gel. Because DNA is a negatively charged molecule, it will migrate toward the positive electrode. Also, because of the construction of the gel matrix, smaller pieces of DNA will move through the matrix more rapidly than will larger pieces of DNA. Thus, fragments can be separated based on their size. Two major types of gels are used for the separatin of DNA fragments, agarose and polyacrylamide. Agarose has the advantages of being easy to use, relatively cheap, and non-toxic. It has the disadvantage of not having great resolving power. In other words, pieces of DNA that are very similar in size usually cannot be separated in an agarose gel. Polyacrylamide has the advantage of exhibiting extraordinary resolution; pieces of DNA that differ in size by a single basepair can be separated in a polyacrylamide gel. Polyacrylamide has the disadvantages of being difficult to use, relativle expensive, and toxic. Primarily, polyacrylamide gel electrophoresis of DNA is used for DNA sequencing. Agarose gel electrophoresis is used for most other applications.
Several factors affect the mobility
of DNA fragments through the gel. In addition to the size of the DNA, these
factors include the voltage used, the type of buffer employed, and the concentration
of the gel matrix. Today, we will investigate the last of these factors, using
a modified version of an experiment originally reported by W. Clark and K. Christopher
(Tested Studies for Laboratory Teaching, 22:81-99) .
Practice preparing agarose gels
Determine the effects of agarose concentration on DNA migration
Review methods for calculating DNA sizes
0.9M boric acid
20 bp DNA ladder (BioRad)
100 bp DNA ladder (BioRad)
500 bp DNA ladder (BioRad)
Each group will prepare one gel of either 0.5%, 1.0%, 1.5%, or 2.0% agarose (w/v). Coordinate with the other groups so that all four gel concentrations are made.
1. Prepare 500ml of 10x TBE. NOTE: you will use this solution throughout the semester.
2. Weigh out the appropriate amount of agarose to make 50ml of the desired gel solution and add it to a 125ml flask.
3. Add 50ml 0.5x TBE to the flask and swirl gently to mix the TBE and agarose.
4. Microwave the flask until the agarose is completely melted.
5. Allow the mixture to cool slightly (until you can pick up the flask comfortably).
6. Add 5uL of ethidium bromide to the mixture. NOTE: ethidium bromide is a known mutagen. Wear gloves!
6. Pour the melted agarose into a gel rig, insert the comb, and allow to harden (approx. 30 minutes).
7. Remove the comb.
1. Carefully slice off a three well section of
your gel. Try to make the cut as straight as possible.
2. In a single gel mold, piece together sections of each gel to make a 0.5%, 1.0%, 1.5%, and 2.0% composite gel. Be careful handling the 0.5% gel. This gel will be very fragile.
3. Place the gel mold in the electrophoresis apparatus and cover the gel with 0.5x TBE.
4. To one well of each section, add 5ul of 20 bp DNA marker + dye.
5. Repeat step 4, this time adding the 100 bp and 500 bp DNA markers to other wells in each section.
6. Connect the electrodes from the gel box lid to the power supply.
7. Run the gel at approximately 80 volts for about 45 minutes.
8. Observe the gel under UV light.
What effect(s) did agarose concentration have on your results?
How would this information be of use to a researcher?
What role does the buffer play?
What would happen if you used water instead of buffer?