Separation by Gel Electrophoresis Using Different Agarose Gel Densities

Advanced Preparation

• Students should have poured their gels the previous day.
• Buffer: You will need to make up approximately 500 mls for each electrophoresis chamber. 8,000 mls of 1XTBE buffer should be enough for 16 chambers. You can reuse the 1XTBE buffer from the chambers so long as it is not badly contaminated by any student team. If contaminated, discard and replace with fresh 1XTBE buffer. Never use buffer that has been used in a chamber for making gels. Gels require fresh buffer.
• Tip boxes: Be sure that all the micropipette tip boxes are filled. These tips do not have to be kept sterile so its OK to handle them without gloves. This task is another one that can be done by your after school students or student service person.
• Dye kits: The dye kits need to be ordered several weeks in advance. They can be purchased from Ward's Natural Science Establishment catalog # 36W5254. These dyes are very inexpensive and can be used for several labs. The kit contains the following: Bromphenol Blue, Janus Green, Orange G, Safranin O, Xylene Cyanol and a tube of some mixture of these 5 dyes. Each tube contains approximately 1ml of dye. You will have to transfer 100µls of each dye to microcentrifuge tubes (1.5ml) and make a set for each team. Place each set of dyes in the microcentrifuge tube rack and place at each lab station. Since the exercise requires 10µls of each dye per gel, one set is sufficient to supply a team of 2 students for up to seven periods.
• Estimated time to label microcentrifuge tubes and transfer dyes is 30-45 minutes. Estimated time to make up 10,000 mls of 1XTBE buffer is 30 minutes. Total estimated prep time 11/2-2 hours.

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
2. Dye set (6 tubes)
3. Power supply
4. Pipette tip box
5. Micropipettor (1-20µl)
6. Waste container
7. Large funnel
8. 1X TBE buffer solution
9. 1/2 inch wide masking tape
10. Student poured gels from previous lab
11. Microcentrifuge tube rack
12. Colored pencils

Recipes for Consumables

1XTBE buffer is made by diluting 100 mls of 10XTBE stock solution in 900 mls of distilled water.

Procedure

1. Place the agarose gel tray into the electrophoresis box as directed by your teacher.
2. Add enough 1XTBE 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. Since this is the first time students will be placing the gel and 1XTBE buffer into the electrophoresis chamber, you should model how to do this step and then you need to check every team. Most students will not put enough 1XTBE buffer into the box and therefore not get the gel wells completely full of buffer. You must be sure that the wells are full of buffer but the level of the buffer is just high enough to barely cover the gel. Another common difficulty is that the gel tray and gel might float, pressing down on the edges of the gel tray until it sits down on the platform in the gel chamber will solve this problem.
3. Dial 10µl on the digital pipette and load six wells with 10µl 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 Loading the gel should not be a problem, however, you may wish to quickly review the micropipette and its use. Special attention to which stop for drawing up and which stop for expelling are probably the most important items to review. Which well is to be lane one is the students choice, just be sure that it is the outermost well on either side of the gel. Then they can load the wells in sequence according to the procedure.
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. Closing the electrophoresis chamber and connecting it to the power supply deserves special attention. Be sure that the lid goes on black lead wire to black electrode and red lead wire to red electrode. Also, be sure that the electrophoresis chamber is positioned where you want it on the table. Once the chamber has been connected to the power supply and the power has been turned on, the student is NOT to touch, or handle the chamber. Check the lead wires from the lid of the chamber to be sure that they have been plugged into the correct receptacles of the power supply. Black to black and red to red and that the wires are plugged into receptacles that are next to each other. Once you have checked all of this at a lab station, you can turn on the power supply and start the experiment running. Set the power supply to a constant 150 volts and use this as the running voltage, then set the run time for 20 minutes. I recommend you do this as the teacher. Any mistake in voltage or run time will invalidate class results. Be sure that the students remove the black and red lead wires of the electrophoresis chamber lid from the power supply BEFORE they remove the lid from the chamber.
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. Have students place their gel directly onto the grid, with the wells over the dark center line. Each line is 2 mm.
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.

Analysis

1. Name the dyes that moved to the positive end of the gel. Bromphenol Blue, Orange G, Xylene Cyanol. What is their electrical charge? Negative. (Note two are monosodium salts.)
2. Name the dyes that moved to the negative end of the gel. Janus Green, Safranin O. What is their electrical charge? Positive. (They are both salt of the anion chloride.)
3. DNA has a phosphate group for each unit. A. What is the formula and charge on a phosphate group? PO₄^3- B. What direction will DNA move (toward which electrode). Toward the positive electrode.
4. If you wanted DNA to move farthest, would you place the loading wells at the positive end, negative end, or in the middle. Place it at the negative end of the gel.
5. Which dye is most likely the smallest molecule? SEE CHART BELOW
6. List the dyes in order of increasing molecular size. SEE CHART BELOW
7. How does your answer in 6 relate to the diffusion of gas lab we did earlier? GENERALLY, THE SMALLER GASES MOVE FASTEST
8. GRAPH. 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. GRAPHS WILL VARY. SOME DIES RESPOND TO THE DIFFERENT GEL DENSITIES WHILE OTHERS DO NOT. IT SHOULD BE NOTED THAT AS THE GELS GET MORE DENSE, SOME DYES MIGRATE A SHORTER DISTANCE.
9. What type of relationship exists between distance and gel density (inverse, direct, exponential) INVERSE
10. How far do you think dye #1 would move in a 1.5% gel density. INTERPRET GRAPH RESULTS.

Reference Chart