Separation by Gel Electrophoresis

Using Different Agarose Gel Densities

Introduction

Like the digital micropipetor, gel electrophoresis is a special technique that molecular geneticists have developed in order to further research in biotechnology. The purpose of this technique is to separate pieces of the DNA molecule by molecular size and shape.

You may recall from other science classes that paper chromatography was used to separate different molecules within a mixture (black ink, leaf pigments). In that method, differences in the molecular weight and solubility of the molecules caused the molecules to rise up the paper at different rates eventually resulting in their separation. Separation by electrophoresis differs from chromatography in that the DNA molecules are moved by an attraction to an electric charge and that the DNA 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, and the density of the gel through which the DNA moves. Since DNA molecules all have the same charge, 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.

Objectives

* Students will show proper technique in the use of gel electrophoresis equipment.

* Students will determine the effect of a molecule's electric charge on its movement through an agarose gel.

* Students will determine the effect of a molecule's size on its movement through an agarose gel.

* Students will show how electrophoresis can separate different molecules from a mixture.

* Students will determine the effect of gel density on the movement of dye macro-molecules.

Materials

1. Electrophoresis chamber 6. Dye set(6 tubes)

2. Power supply 7. Pipette tip box

3. Pre-poured agarose gel(2%) 8. Waste container

4. Micropipettor (1-20ul) 9. 1X TBE buffer solution

5. Large funnel

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 being careful not to let the gel slide off the tray or puncturing the gel with your fingers.

3. Dial 10ul on the digital pipette and load six wells with 10ul of dye according to the following pattern. 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 Safranin O

5 Xylene Cyanol

6 Dye mixture

4. Place the cover onto the gel box being 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.

6. After all teams are plugged into the power supply, have your teacher check the set up. Once okayed, plug in the power supply to the electrical socket and set it according to your teacher's instructions. DO NOT take the cover off the gel box 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

1. Label the diagram below with the following:

A. The positive and negative ends of the gel ()

B. Lane number 1-6. This must correspond to the way you loaded the gel in step 3 of the procedure.

C. Direction of current flow through the gel (direction negatively charged electrons move)

2. After you record the above data, place the Gel Measurement Grid located on the last page of this lab.

3. Record the distance dyes 1-5 traveled on the data table provided on the next four pages. Be sure to use the correct table which corresponds to the Gel % that you used.

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

5. Place your information in the table on the overhead or on the board as directed by your teacher.

6. Record the data from other groups onto the tables.

( ) ( )

Gel Density 1%

Lane                Dye                 Direction Dye       Distance Dye        
                                        Moved               Moved (mm)          
1                                                                               
1                                                                               
1                                                                               
Average of 1                                                                    
                                                                                
2                                                                               
2                                                                               
2                                                                               
Average of 2                                                                    
                                                                                
3                                                                               
3                                                                               
3                                                                               
Average of 3                                                                    
                                                                                
4                                                                               
4                                                                               
4                                                                               
Average of 4                                                                    
                                                                                
5                                                                               
5                                                                               
5                                                                               
Average of 5                                                                    
                                                                                

Gel Density 2%

Lane                Dye                 Direction Dye       Distance Dye        
                                        Moved               Moved (mm)          
1                                                                               
1                                                                               
1                                                                               
Average of 1                                                                    
                                                                                
2                                                                               
2                                                                               
2                                                                               
Average of 2                                                                    
                                                                                
3                                                                               
3                                                                               
3                                                                               
Average of 3                                                                    
                                                                                
4                                                                               
4                                                                               
4                                                                               
Average of 4                                                                    
                                                                                
5                                                                               
5                                                                               
5                                                                               
Average of 5                                                                    
                                                                                

Gel Density 3%

Lane                Dye                 Direction Dye       Distance Dye        
                                        Moved               Moved (mm)          
1                                                                               
1                                                                               
1                                                                               
Average of 1                                                                    
                                                                                
2                                                                               
2                                                                               
2                                                                               
Average of 2                                                                    
                                                                                
3                                                                               
3                                                                               
3                                                                               
Average of 3                                                                    
                                                                                
4                                                                               
4                                                                               
4                                                                               
Average of 4                                                                    
                                                                                
5                                                                               
5                                                                               
5                                                                               
Average of 5                                                                    
                                                                                

Gel Density 4%

Lane                Dye                 Direction Dye       Distance Dye        
                                        Moved               Moved (mm)          
1                                                                               
1                                                                               
1                                                                               
Average of 1                                                                    
                                                                                
2                                                                               
2                                                                               
2                                                                               
Average of 2                                                                    
                                                                                
3                                                                               
3                                                                               
3                                                                               
Average of 3                                                                    
                                                                                
4                                                                               
4                                                                               
4                                                                               
Average of 4                                                                    
                                                                                
5                                                                               
5                                                                               
5                                                                               
Average of 5                                                                    
                                                                                

Analysis

1. Name the dyes that moved toward the positive end of the gel electrophoresis box.

What is their electrical charge?

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

What is their electrical charge?

3. DNA has a phosphate group for each unit.

A. What is the formula and charge on a phosphate group?

B. What direction will DNA move (toward which electrode)

4. If you wanted to have a maximum migration distance for DNA, would you place the loading wells at the positive end, negative end, or in the middle.

5. Which positively charged dye appears to be the smallest molecule?

6. List the positively charge dyes in order of apparent increasing molecular size.

7. Which negatively charged dye appears to be the smallest molecule?

8. List the negatively charge dyes in order of apparent increasing molecular size.

9. How does your answer in 6 relate to the diffusion of gas lab we did earlier?

10. On a separate piece of graph paper plot the average distance dyes 1-5 moved on the y axis against gel density on the x axis. You will have 5 lines.

9. What type of relationship exists between distance and gel density (inverse, direct, exponential)

10. How far do you think dye #1 would move in a 1.5% gel density.

Conclusion

The molecular formula of each dye is given in the chart below.

1. Using a periodic table, determine the molecular mass of each compound, and place it in the appropriate box in the chart.

 Lane        Compound                  Formula                    Mass         
1       Bromphenol Blue     C19H10Br4O5S                                       
2       Janus Green         C30H31N6Cl                                         
3       Orange G            C16H10N2O7S2Na                                     
4       Safranin O          C20H19N4Cl                                         
5       Xylene Cyanol       C27H31N2O6S2Na                                     
6       Dye mixture         Some or all of the above                           

2. What conclusion can you draw when you compare the formula of dies 2 and 4, with the direction they migrated on in the gel box?

3. What conclusion can you draw when you compare the formula of dies 3,5 with the direction they migrated on in the gel box?

4. Is mass the only determinant of how far a molecule will move? What evidence do you have for your answer?

5. Does mixing the dies change their properties? What evidence do you have for your answer?

6. Which dye must have a charge of more than 1? Why would you make that conclusion?