Materials Included In Kit

Additional Materials Required

Prelab Preparation

Preparation of 1X electrophoresis buffer

• Add 8 mL of 50X TAE buffer to 392 mL of tap water (distilled water is preferable) in a 500-mL Erlenmeyer flask. Pour into bottles, seal with a cap, label and store in a refrigerator. Note: Prepare enough buffer solution to allow each group to cover the gel in the chamber to a depth of about 2 mm. Depending on the type of electrophoresis units being used; the amount of buffer needed could be as much as 250 mL/chamber.

Preparation of agarose gels

Note: To save time, cast the gels the day before the lab, using the procedures that follow:

1. Loosen the cap on the bottle of agarose.
2. Place in a hot water bath until completely melted and liquefied or place the bottle in a microwave oven and heat on high at 1-minute intervals until liquefied.
3. Use protective gloves to remove the bottle. To prevent damage to the casting trays, allow the agarose to cool slightly (2 minutes maximum) before pouring.
4. Carefully pour the melted agarose into the assembled casting tray with the well-forming comb in the middle of the gel box. Only add enough agarose to equal the height of the indentations in the well-forming comb.
5. Allow the gel to sit undisturbed at least 20 minutes until completely set. (The set gel will appear opaque and somewhat white.)
6. Once the gel is thoroughly set, carefully remove the well-forming comb by rocking it gently from side to side and then pulling it upward. Remove the end dams also.
7. Slide each gel into a separate zipper-lock bag and then refrigerate.

Preparing “known” dye samples for each group

Label microcentrifuge tubes with numbers 1–6, using a fine-point marker. Prepare six tubes for each group by adding 20 μL of dye solution to each microcentrifuge tube. Note: The needle point end of each pipet included in the kit holds 10 μL.

Preparing “unknown” dye samples for each group

All of the dye solutions in this kit have been prepared to allow different combinations of dyes to be used to make unknowns. Add 10 μL of two or three dye solutions to a tube. Then remove 20 μL of the mixture and place in microcentrifuge tube 6 for each group. Note: If making an unknown with three dye solutions, after mixing, remove and discard 10 μL. Do not mix more than three dyes together.

Safety Precautions

Be sure all connecting wires, terminals and work surfaces are dry before using the electrophoresis units. Electrical Hazard: Treat these units like any other electrical source—very carefully! Do not try to open the lid of the unit while the power is on. Exercise extreme caution in handling the dyes; they will all readily stain clothing and skin. Wearing chemical splash goggles and gloves is strongly recommended. Wash hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. All solutions used in this lab may be disposed of down the drain using copious amounts of water according to Flinn Suggested Disposal Method #26b. Used gels may be disposed of in the regular trash according to Flinn Suggested Disposal Method #26a. All microcentrifuge tubes may be rinsed with distilled water and reused.

Lab Hints

• Enough materials are provided in this kit for 32 students working in groups of four or for 8 to 10 groups of students. This laboratory activity can reasonably be completed in one 50-minute class period provided that the Prelab Activity is completed and discussed and all the gels have been pre-poured and are ready to use.
• Before leaving class, students must complete the distance measurements for each dye sample. Although the gels may be saved in individually-labeled zipper-lock bags and stored in the refrigerator, the dyes will continue to diffuse within the gel and accurate measurements will not be possible the next day.
• The sample data included with the kit were collected after 28 minutes at 70 V; however, the voltage on the electrophoresis units may be increased. When run at 125 V, only 12 minutes were required to separate the dyes. At a higher voltage, constant monitoring is critical to ensure the dyes are not pulled all the way through and out of the gel.

Teacher Tips

• Demonstrate the need for and importance of using filters, in general, to separate mixtures by adding sand to water. Pour the mixture through a strainer into a beaker and then ask how many students would be willing to drink the water? What could be used to filter the water better?

• The procedures have been written assuming that all the gels will be pre-poured and ready for each group. If you want student groups to melt and pour their own gels, have them follow the instructions for Preparation of agarose gels in the Prelab Preparation section and add at least one extra day to complete the lab.
• For indefinite storage of dye solutions, wrap bottles in aluminum foil and store at room temperature. Extra, unused, diluted and undiluted buffer may be stored in capped, labeled containers in a refrigerator.
• Help students understand the difficulty in making predictions about the behavior of molecules they cannot see by setting up five microscopes in the room with one drop of each dye on a slide. Place a cover slip over the drop to help prevent drying out, then after locating the drop, rotate the 10X objective into position. Students should only need to adjust the fine focus while viewing each dye droplet. Have them write down the name of each dye and briefly describe the color they observe and any other distinguishing characteristics.
• The diagrams of the structural formulas shown for each of the organic dyes do not include the oppositely charged or counter ions for the sake of simplicity.

Answers to Prelab Questions

1. Predict the direction of migration for each of the known dye samples.

Student answers will vary.

2. Which dye do you predict will move through the gel the fastest? The slowest?

Student answers will vary.

3. Write a brief function for each of the parts used in gel electrophoresis:

1. Agarose gel

   The agarose gel is the microscopic “strainer” that separates big molecules from small ones because of the size of the pores within the agarose.

2. Electrophoresis buffer

   It is the liquid conductor that carries the electric current.

3. Wells in the gel

   They hold the sample that will travel through the gel.

4. Electric current

   It provides the force that moves the sample through the gel.

4. List one important safety precaution that must be followed when performing any type of gel electrophoresis.

Answers may include: Ensure work surface, connecting wires and terminals are dry. Do not try to open the lid while the power is on.

Sample Data

Answers to Questions

1. Which dye(s) traveled the farthest? (Use data to support your answer.)

Student answers may vary but it should be Alizarin Red S, which traveled 14 mm.

2. How did the results differ from your predictions regarding migration direction, which dye(s) would travel the fastest, and which dye(s) would travel the slowest? Be specific.

Student answers will vary.

3. Why didn’t all the dyes travel the same distance or the same direction from the wells? Explain your answer.

Because all the dyes had different molecular weights, charges and shapes. The smallest, more linear dye seemed to travel faster than the larger, more branched dyes.

4. Why did the two positively charged dyes travel almost the same distance? (Use a mathematical calculation to support your answer.)

Malachite Green has a MW of 329, Safranin O has a MW of 315 therefore they have difference in mass of only 4% (calculated by dividing the difference by 329). This may account for the similarity in distance traveled.

5. Of the three negatively charged dyes, why do you think the “heaviest” was the second fastest dye? (Hint: Look closely at the structural formula.)

Student answers will vary but may include: Orange G has two negative charges making it perhaps move faster toward the positive electrode. Its structure also appears more linear.

6. List the dyes that were used in your “unknown” dye sample.

Student answers will vary depending on the dyes used to make the unknown.

7. Write one reasonable explanation to support the answer given for Question 6.

Student answers will vary but should include something about matching the colored bands of the “unknown,” in both charge and migration distance, to the bands of the known dyes.

8. Explain the basis for the “speed” rankings given to the known dye samples in the data table.

The speed rankings are based on the distance each dye migrated from the well regardless of the migration direction.

9. List three errors that could affect the outcome of any gel electrophoresis procedure.

a. Not placing the sample deep enough into the well or not placing enough sample into the well.
b. Puncturing the well with the pipet tip causing the dye to actually run below the gel.
c. Connecting the wires to the power supply or chamber incorrectly.
d. Contaminating pure samples by using the same pipet tip in different samples.
e. Not recording which sample went into which well according to their charge, size and shape.

10. Briefly summarize how gel electrophoresis is used to separate molecules.

Student answers will vary but should include how this technique involves the use of a gelatin-like material that acts as molecular filter paper. When samples are placed in wells within the gel and the electricity turned on, the resulting electric field causes the molecules, which make up the samples, to separate according to their charge, size and shape.

References

An Introduction to Electrophoresis. Laboratory Activity 2. American River College Biotechnology Program, Sacramento, CA, 2002.

All About Samples! Genetic Science Learning Center—Eccles Institute of Human Genetics, University of Utah, Salt Lake City, UT.

Introduction

How can a mixture of molecules, too small to be seen with even a high-powered microscope, be separated from one another? Such was the dilemma facing scientists until the development of a process that has now become standard in many laboratories worldwide—gel electrophoresis. Laboratories rely heavily on this proven and reliable technique for separating a wide variety of samples, from DNA used in forensics and for mapping genes, to proteins useful in determining evolutionary relationships.

Concepts

• Biotechnology

• Biological molecules
• Electrophoresis

Background

As it became more important for substances to be identified by their molecular structures rather than by direct observation, scientists were challenged with how to separate and isolate molecules from one another. Over the years, separation techniques, such as distillation, crystallization, liquid and gas chromatography and atomic absorption, were developed to separate and identify molecules. However, biological molecules like proteins and DNA, are very large and sensitive and many of the aforementioned separation techniques are not appropriate.

In 1950, a scientist named Oliver Smithies invented gel electrophoresis. The process involves applying an electrical current to a gelatin-like substance containing biological samples. When mixtures of materials are placed within the wells or openings of the gel and an electric current is applied, the molecules travel through the gel and separate from one another according to each molecule’s charge, size and shape. The gel, called agarose, is made from an extract of red algae. It acts as a type of molecular strainer or sieve and prevents all the molecules from moving too quickly. However, like pouring vegetable soup through a strainer, smaller molecules typically move through the gel at a faster rate than larger ones. When the current is turned off, all the molecules are stopped within the gel. If a dye is added to the samples placed in the wells, individual groups of molecules can be identified by a distinct, colored band within the gel. When the distance each band (group of molecules) traveled is measured and compared to the other colored bands within the gel, the measurement can readily distinguish smaller, molecular weight molecules from larger molecules. The Biotech revolution of today really began with the refinements and adjustments made to this dependable separation technique.

Experiment Overview

The purpose of this activity is to demonstrate the separation technique known as gel electrophoresis. This process will be used to identify dye samples by charge, molecular mass and shape. The information will also be used to identify the composition of an “unknown” mixture of dyes.

Materials

Safety Precautions

Be sure all connecting wires, terminals and work surfaces are dry before using the electrophoresis units. Electrical Hazard: Treat these units like any other electrical source—very carefully! Do not try to open the lid of the unit while the power is on. Exercise extreme caution in handling the dyes; they will all readily stain clothing and skin. Wear chemical splash goggles, chemical-resistant gloves and apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

1. Depending on the type of electrophoresis units available, assemble the unit according to the teacher’s instructions with the end dams in place.
2. Place the electrophoresis unit in a horizontal position on a level table or countertop.
3. Place the “Gel Drawing Worksheet” on the counter horizontally next to or below the electrophoresis unit. The small rectangles on the paper correspond to the wells in the gel. Number the wells on the paper from well 1 at the top to well 6 at the bottom (see Figure 1).
4. Gently slide a gel from a zipper-lock bag into the casting tray and between the end dams. Pour enough electrophoresis buffer (1X) into the unit to just cover the entire gel surface. Remove the end dams.
5. Withdraw 10 μL of dye from each microcentrifuge tube by filling only the needle tip of the pipet. Note: Fill the tip by squeezing the pipet just above the tip, not the bulb. Use a clean pipet for each dye sample to avoid contaminating the pure samples. Dispense a sample of each dye into a different well in the gel. Record the name and well number of each dye in the data table.
6. Place the lid on the chamber and connect it to the power supply according to teacher instructions. Allow electrophoresis to proceed for 20–25 minutes at 70 V before turning off the power.
7. When the power is off, remove the cover and with the help of a ruler, carefully remove the gel from the chamber and put it on a piece of paper towel.
8. Measure the distance each dye migrated in millimeters (mm). This distance should be measured from the side of the well closest to the direction the dye traveled to the leading edge of each colored band. Record this number in the data table as Migration Distance (mm).
9. Use the Gel Drawing Worksheet to make an accurate drawing of the bands in the gel. Colored pencils may be helpful in identifying each band in the drawing.
10. Record the charge for each group of molecules by noting to which pole (±) each dye sample was traveling when the electricity was shut off.
11. Use the recorded Migration Distances to complete the last column of the data table by ranking the five known dye samples from fastest (well 1) to slowest (well 5).
12. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

10666_Student1.pdf