THE SEPARATION OF DYE MOLECULE

THE SEPARATION OF DYE MOLECULES USING GEL ELECTROPHORESIS LAB
Adapted from Fred Hutchinson Cancer Research Center Science Education Partnership Lab

BACKGROUND INFORMATION:

Agarose gel electrophoresis is a method to separates molecules on the basis of charge, size and, shape. The ionic strength (charge), viscosity, and temperature of the medium also affect the mobility or migration rate of molecules in the electric field. The method is particularly useful in separating charged biomolecules such as DNA, RNA, and proteins. Agarose gel electrophoresis possesses great resolving power, yet is relatively simple and straightforward to perform. The gel is made by dissolving agarose powder in a buffer solution by boiling the solution. The solution is then cooled and poured into a mold where it solidifies. The gel is then submerged in a buffer-filled chamber which contains electrodes. Samples are prepared for electrophoresis by mixing them with a dense solution such as glycerol or Ficoll. This makes the samples denser than the electrophoresis buffer. These samples can then be loaded with a micropipet or transfer pipet into the wells that were created in the gel by a template called a comb. The dense samples sink through the buffer and settle in the wells. A direct current power supply is connected to the electrophoresis apparatus and current applied. The buffer in the gel chamber completes an electric circuit between the electrodes. Charged molecules in the sample enter the porous gel through the walls of the wells. Molecules having a net negative charge (anions) migrate toward the + electrode (anode, red) while positively charged molecules (cations) migrate towards the negative electrode (cathode, black). The higher the applied voltage, the faster the molecules travel. The buffer salts serve to make the water a better conductor of electricity and to control the pH. The pH is important to the charge and stability of many types of molecules.

Agarose is a polysaccharide derivative of purified agar. The agarose gel contains microscopic pores which act as molecular sieves. The sieving properties of the gel influence the rate at which molecules migrate. Smaller molecules move through the pores more easily than larger ones. Molecules can have the same molecular weight and charge but different shapes. Molecules having a more compact shape (sphere is more compact than rod) can move more easily through the pores. Given two molecules of the same molecular weight and shape, the one with the greater charge will migrate faster. The factors of charge, size and shape interact with one another to various extents depending on the molecule's structure and composition, buffer conditions, gel concentrations and voltage.

OBJECTIVES:

Make and load the wells of an agarose gel.
Electrophorese samples in the gel and interpret the results.

Identify and manipulate variables involved in separating biological dyes
Design and carry out a simple electrophoresis experiment.
Explain the theory behind separation of molecules by electrophoresis.

PRE-LAB QUESTIONS:

1. On what basis does electrophoresis separate molecules?
2. What are three types of charged biomolecules that electrophoresis is particularly useful in separating?
3. What is the gel made of?
4. In what is the gel submerged?
5. What substance is used to make the samples denser?
6. Why do the samples need to be made more dense than the buffer?
7. What is the purpose of the buffer in the gel chamber?
8. Which way will negatively charged molecules migrate?
9. What is the purpose of the buffer salts?
10. Why is it important for the buffer to maintain pH?
11. What characteristic of agarose makes it useful for electrophoresis?
12. What happens to molecules having the same charge and weight when run through an agarose gel?
13. List variables that influence the way the charge, weight and shape of molecules interact with each other during electrophoresis.
14. DNA in solution is negatively charged; if you ran a sample of DNA, what would you predict about its migration?

PROCEDURE:

1. Pour hot, liquid gel into the gel mold and add the comb, let set while class goes through the pre-lab questions. When gel appears to have solidified, remove comb and mold ends.
2. To prepare the electrophoresis apparatus: place the gel into the gel box. Add 1X TAE buffer to just cover the gel about 1-2mm (~125ml).
3. Before loading samples, be sure your gel box is near the power supply.
4. Obtain samples by pipetting 20μl (this is 15μl to load in the well plus a little extra) of each dye sample from the class supply into the appropriately well that you have labeled in your diagram. You will be using methyl red, methyl orange, crystal violet, methylene green, methylene blue, safarin and two unknowns that are a mixture of your knowns.
5. When all 8 samples are loaded, close the lid on the gel box. Student groups will share the power supplies. Check your power supply first: the main power switch on the power supply must be turned off when gel boxes are being connected to the power supply. Connect the electrodes to the gel box and the power supply connecting red to +(anode) and black to — (cathode). Turn on power supply and set it to 100 volts. Check the milliamp output. Electrophorese for a total of 10 min. If power supply beeps, turn down the voltage.
6. Observe and record the changes that you see happening after about 5 min on your record sheet. Toward which electrode did the greater number of samples run?
7. After the 10 min run, turn off the power. Unplug the electrodes and open gel box. Lift out the gel deck and gel and tip slightly to pour off excess buffer. Place them on the lab table on a paper towel and wipe off excess buffer. Record your results on your record sheet.
8. Measure the distance that each colored spot traveled on the gel. Measure in mm from the lower edge of the well to the center of the spot. Write your data on your record sheet.
9. Wrap up the gel in plastic wrap, label, and freeze for future reference if needed.

RESULTS:

Well Number	Name of the Dye	Distance Traveled (in mm)	Pole (+/-)	Comments and Observations
1
2
3
4
5
6
7
8

OBSERVATIONS:

Before

After

ANALYSIS QUESTIONS:

1. What was the charge of each molecule in the wells?

2. How do the sizes compare between the molecules from the 8 wells?

3. Did any substance you put in the wells have more than one type of molecule in it? How do you know?

4. If you were asked to improve the separation of these molecules, what are some of the variables you could modify in your own experiments?

5. Write two questions you have about this process and the results. Indicate how you might generate answers to these questions through experimentation.