ElectrophoresisB

DYE ELECTROPHORESIS

Introduction

Like the digital micropipet, gel electrophoresis is a special technique that molecular geneticists have had to develop in order to further their research in biotechnology. The purpose of this technique is to separate pieces of the DNA molecule by molecular size/shape and electrical charge.

Prior to the development of electrophoresis, chromatography was used to separate different molecules within a mixture such as black ink and leaf pigments. In that method, differences in the molecular weight and solubility of the molecules caused the molecules to rise at different rates eventually resulting in their separation. Separation by electrophoresis differs from chromatography in that the molecules are moved by an attraction to an electric charge and they must diffuse through a porous agarose gel. A good analogy of a gel is a sponge, if you can imagine how large and small particles would pass through a sponge at different rates according to particle size/shape versus the size of the holes in the sponge. The rate of molecular movement depends upon molecule size/shape and the direction of movement depends on the electric charge (+ or -) of the molecules. DNA molecules all have the same charge, therefore, they all move in the same direction. Today, however, you will be separating different color dyes, some have a positive charge and others a negative charge.

With buffer in an electrophoresis chamber, when you can add a pH indicator (e.g. cabbage juice), the buffer will change color, based on the positive and negative charges that are present at each electrode. These color changes demonstrate that acids have a predominance of positive ions (H⁺) while bases have a predominance of negative ions (OH^-).

Objectives

* The student will use the gel electrophoresis equipment.

* The student will demonstrate how charge and size affect a molecule's movement through a gel matrix.

* The student will use the principles of electrophoresis to separate different molecules from a mixture.

Materials

1. Electrophoresis gel box 7. 1X TBE buffer solution

2. Electrophoresis gel tray and comb 8. Dye set (6 tubes)

3. Power supply 9. Pipet tip box

4. 2% agarose gel 10. Waste container

5. Micropipet (1-20 ul) 11. Large funnel

6. 1/2 inch masking tape

Procedure

1. Place the agarose gel tray into the electrophoresis box as directed by your teacher.

2. Add enough 1X TBE buffer into each side of the box until the gel is barely covered. If air bubbles form under the gel tray, gently lift one edge of the tray to release the air. Be careful not to let the gel slide off the tray or puncture the gel with your fingers.

3. Dial 10 ul on the digital micropipet. Load six wells with 10 ul of dye according to the following pattern. Lane #1 is the outermost well on the side of the box with the black electrode. (Be sure to change tips between each dye so as not to contaminate the dye tubes!)

Lane # Dye

1 Bromphenol Blue

2 Janus Green

3 Orange G

4 Safarin O

5 Xylene Cyanol

6 Dye mixture

4. Place the cover onto the gel box. Be sure the wire plug-ins match black to black and red to red.

5. Be sure that the power supply is unplugged before connecting the gel box wires to it. Match the red wire to the red receptacle and the black wire to the black receptacle. Be sure you use the receptacles that are next to each other.

6. After all teams are plugged into the power supply, have your teacher check the set up. Then, connect the power supply to the electrical socket and set it according to your teacher's instructions. DO NOT remove the gel box cover while the electric current is on.

7. For twenty minutes observe the movement of the dyes within the gel as the electric current passes through the buffer and gel.

Data

Label the diagram on the next page with the following:

1. the positive and negative ends of the gel ( )

2. lane numbers 1-6; this must correspond to the way you loaded the gel in step 3 of the procedure

3. direction of current flow through the gel (this is the flow of electrons which are negatively charged)

4. using colored pencils, record the direction and distance moved by each dye

Clean Up

1. Turn off the power supply then unplug it.

2. Discard the gel. Thoroughly rinse the tray with tap water to remove the 1X TBE buffer solution.

3. Using the large funnel pour the 1X TBE buffer solution into its bottle.

4. Rinse the gel box with tap water. DO NOT wipe the inside of the box with paper towels. Instead, turn it upside down on paper towels to dry. 5. Rinse the box cover with tap water. Let it dry with the gel box.

Analysis

1. Name the dyes that moved to the positive end of the gel.

What is their electrical charge?

2. Name the dyes that moved to the negative end of the gel.

What is their electrical charge?

3. Explain how you knew which dyes from questions 1 and 2 had which electrical charge.

4. Which dye is most likely the smallest molecule? Why?

5. List the dyes in order of increasing molecular size.

6. How can you tell that the dyes in the mixture are actually the same dyes found in other lanes? (color isn't the answer)

7. DNA is a deoxyribose sugar and phosphate polymer (this means these units are repeated over and over hundreds of times). The phosphate units (PO4^3-) give all DNA molecules a negative electrical characteristic. In what direction would you expect pieces of DNA to move during electrophoresis? Why?

8. Therefore, when placing a gel into the electrophoresis box, the wells should be closest to which electrode? Why?

Conclusion

Explain the principles involved in the separation of molecules by gel electrophoresis.