NAME: ________________________________
PREPARING ANAGAROSE GEL FOR ELECTROPHORESIS OF DNA
PURPOSE
To prepare a 1% agarose gel for electrophoresis of DNA samples. To understand the process of electrophoresis for the separation of DNA fragments based on size and why this is important to biotechnology.
INTRODUCTION
Agarose is a complex carbohydrate polymer extracted from seaweed that can be melted and dissolved in hot water. This mixture when heated produces a transparent solution where the carbohydrate chains float freely. The melted solution is poured into a casting tray and allowed to cool and harden into a gel, a semi-solid matrix much like “jello" where the carbohydrate chains form a tangled web of fibers. The gel can be used to separate DNA fragments by size (length) using a process known as gel electrophoresis. DNA molecules are highly negatively charged because of the phosphate groups of the sugar-phosphate backbone of the DNA molecule. When an electric field is applied across the gel, DNA molecules will migrate through the gel matrix toward the positive pole. As the DNA strand moves through the gel, it can be slowed down by the tangled web of the agarose chains. Smaller DNA fragments move through the gel more quickly than larger fragments. Fragments of the same size migrate together through the gel, forming bands of DNA. An analyst determines the size of DNA fragments by the migration distance; smaller fragments will move farther through the gel than larger fragments. A DNA sample containing DNA fragments of known size (DNA ladder or markers) is also run on the gel. The sizes of unknown fragments are determined by using the DNA ladder to create a standard curve of size versus distance migrated. Separating DNA fragments is important for the study of individual genes, their structures, and their functions.
Now let’s try to make a gel!
MATERIALS
Latex gloves 2 Gaskets
Agarose powder Gel comb
1X TAE buffer Paper towel
250 mL flask Protective heat gloves or oven mitt
Gel Box Microwave
PROCEDURE
1. Before beginning be sure to glove your hands in order to prevent contamination. Put on safety glasses.
2. Prepare the casting tray by inserting the red rubber gasket into each grooved side of the clean and dry casting tray. Be sure that the gasket is seated into the grooves along their entire length.
3. Place the casting tray horizontally into the gel box so that the gaskets are flush against the sides of the box. It may take a little force to wiggle the casting tray into the box. The casting tray should be sitting flat against the bottom of the gel box.
(It is important that the gasket stay in the groove of the casting tray, if it becomes displaced pull the casting tray out and start the process over.)
4. Measure 0.5 g of agarose powder into a weigh boat and then add to a clean 250-mL Ehrlenmeyer flask.
5. Measure 50 mL of 1 X TAE buffer using a graduated cylinder and carefully pour it into the flask. Do not splash or cause the powder to fly out of the flask.
6. Swirl the mixture gently, without getting very much agarose stuck to the sides of the flask.
7. Loosely cover the flask with a tissue or a paper towel. Place the flask in the microwave and heat for 15-20 seconds on high. CAUTION - the agarose solution can become superheated and may boil violently if it has been heated in the microwave oven. Take great care in handling the flask. Use a hot glove and be careful not to swirl quickly as the flask may boil over and cause a burn. Gently swirl the solution and resume heating for 15-20 seconds. Repeat the process every 15-20 seconds until the agarose dissolves completely. How can you tell that? Well, make sure that you have a crystal clear mixture in your flask. No particles (even clear ones) must be present. Avoid boiling your solution too long because you can lose much your initial volume.
CAUTION: Flask and solution will be HOT! Be sure to use protective heat gloves or oven mitts. Use caution when handling the hot agarose solution to avoid boiling over.
8. Carefully pour the hot solution evenly into the casting tray. Avoid tilting or moving the box after pouring the gel, as this can disturb the gel matrix and limit polymerization. It is important that there are no air bubbles visible in the gel.
(You may use a pipette tip to move any bubbles in the liquid to the side of the casting tray.)
9. Place the comb into the liquid gel by fitting it into the grooves at the top of the casting tray. The comb has two different thicknesses. For this experiment we will use the thickest side of the 10-well comb. This creates wells (little cups) in the gel where DNA samples will be loaded.
10. Allow the gel to cool for 20-30 minutes.
Helpful Hint: If you place your hand underneath the gel box it should be cool to the touch when ready. The gel will also have changed from clear to opaque.
11. Gently remove the comb from the gel by pulling straight up. Be sure to apply even pressure to all parts of the comb so the wells of the gel are not broken.
12. Carefully wiggle the casting tray out of the gel box. Remove the rubber gaskets from the sides of the casting tray.
13. Place the casting tray back into the box, orienting it so the wells are closest to the black electrical lead, the negative pole (annode).
14. Flood the gel box with 250 mL of 1 X TAE Buffer. The gel should be submerged.
You are now ready to load the DNA and run the gel!
QUESTIONS AND CONCLUSIONS
1. Which of the following images represents agarose?
A B C
2. Describe two precautions you take when you make agarose gels.
3. Using reference materials define the following:
a. Polymer:
b. Buffer:
c. Gel:
d. Electrophoresis:
e. DNA: