Introduction to Gel Electrophoresis

Transcription

1 Experiment 1 Introduction to Gel Electrophoresis Gel Electrophoresis is a method used by molecular biologists to study molecular characteristics of many biomolecules. The term electrophoresis is defined as the movement of suspended particles through a medium or matrix under the influence of electricity. Gel electrophoresis serves as a tool providing descriptive data about the charge, shape, size, and composition of molecules based on the distance the components of the sample move. Commonly used with nucleic acids, gel electrophoresis helps life scientists discover new relationships and information about living organisms. In this activity, you will use gel electrophoresis to learn about the composition and relationship of food colorings. You will use a gel matrix of buffered agarose to measure the migration of food coloring molecules when they are subjected to an electric field. This agarose gel is precast into a conveniently sealed cassette that snaps into a compact power base powered by a 12 V adapter, as seen in Figure 1. OBJECTIVES During this activity, you will Use the E-Gel system to perform agarose gel electrophoresis on individual and combinations of common food coloring dyes. Measure the migration distance of dye fronts exposed to an electric field. Determine whether the food coloring dyes are composed of single or multi-colored molecules. Determine the composition of these common food coloring dye samples. MATERIALS Food Coloring Set E-Gel power base and power supply E-Gel cassette Micropipettor (2 20 µl) or fine tipped Beral pipette Micropipette tips Metric ruler Sheet of white paper PROCEDURE 1. Connect the AC power adapter to the E-Gel Power Base. The base should beep twice and the LEDs will flash. 2. Remove the E-Gel cassette from its package and place it into the base by inserting the right edge first and then snapping it into place. The LED will be red. Press and hold the 15-minute button until the base beeps twice, then release the button. This initiates the required two minute warm-up period. At the end of that period, the base will repeatedly beep. To cancel the alarm, press the 15-minute button once. 3. Remove the comb from the E-Gel.

2 Experiment 1 4. Load the E-Gel with food coloring samples: a. Put a clean micropipette tip on the pipetter. b. Obtain the food coloring sample labeled #1 and record the color of the sample in the space provided in the data table below. c. Using the micropipette, draw up 20 µl of sample 1. d. Dispense the 20 µl into the well of lane 1. e. Dispose of the micropipette tip. 5. Follow the procedure outlined in Step 4 for the remaining samples. Be sure to put each numbered sample in its corresponding lane (sample 3 in lane 3, sample 5 in lane 5, etc.). Be sure to use a clean micropipette tip for each new sample to avoid cross contamination of the samples. 6. Using a clean micropipette tip, fill any unused lanes with 20 µl of water. 7. Once you have all lanes filled with either a food coloring sample or water, press the 15-minute button to start the gel run. 8. When the run is complete, the base will beep. Disconnect power from the base and remove the E-Gel.

3 DATA ANALYSIS Introduction to Gel Electrophoresis 1. Place the E-Gel on a white piece of paper. Measure and record the color and distance for each band found in each sample lane. Measurements are made from the lowest edge of each well to the lowest edge of each band. A lane may have more than one band. Lane No. Initial Color Post-Run Band Colors and Distances (Migrated color/mm) Define each of the following terms and explain how they differ in meaning: stains, dyes, fixatives, mordants, colorants, pigments, and fluorochromes. 3. From your results, what general statements can you make about the color composition of the individual food colorings? What can you conclude from your results regarding the physical and chemical nature of food colorings? 4. What might be responsible for one band of color advancing further than another in the gel? 5. What considerations would a producer of coloring agents need to consider before marketing such a product?

4 Experiment 1 6. Compare and contrast the color bands found in lanes 3 and 9. What conclusions can you make about these two samples based on this experiment? 7. Seen in the light of agarose gel electrophoresis, what deductions can be made about the character of the different colored bands? EXTENSIONS 1. Determine the effect that concentration of the food coloring samples has on the results. 2. Compare different brands of food coloring by using gel electrophoresis. 3. Investigate the effects of running the gel for 30 minutes instead of 15-minutes. 4. Investigate the effects of using different percent agarose gels on your results.

5 TEACHER NOTES Introduction to Gel Electrophoresis 1. To prepare each food coloring set: a. Label six 1.5 ml microtubes with the numbers 1, 3, 5, 7, 9, and 11. b. Using standard food colorings of blue, green, red, and yellow, add drops of the following colorings and water to the specified numbered microtubes as listed. This will make enough product to run between 15 and 20 gels. Microtube No. Coloring & No. of Drops No. of Drops of Water 1 Blue Green Red Yellow Blue 1 Yellow 1 Blue 1 Red There are several options available for pipets: A fixed-volume micropipette with 20 µl capacity or a variable-volume micropipette with a range of 2 20 µl works well. Micropipette tips are required with these two units. A thin-stemmed Beral pipette can be used and will transfer between 20 and 25 µl per drop. A thin-stemmed Beral pipette can be modified to be more precise by stretching out the stem. To do this, hold the bulb with one hand, place the thumb and forefinger of the other hand about three centimeters from the bulb on the stem, and gently pull. The stem should extend, forming a thinner diameter. Continue the extension until there is at least a continuous 5 centimeter section of the newer diameter stem. Using a ruler and a pair of scissors, measure from the initiation of the narrower stem closest to the bulb to a point leaving 3 centimeters of narrowed stem attached to the bulb and clip. This 3 centimeter section of the stem will hold approximately 20 µl of sample. 3. E-Gels from this experiment can be reused by other groups doing 15-minute runs of food coloring. You should be able to get three 15-minute runs for each E-Gel.

6 Experiment 1 SAMPLE RESULTS 1. Record of color and distance migration results. Lane No. Initial Color Post-Run Band Colors and Distances (Migrated color/mm) 1 xxxxxx xxxxxx 3 xxxxxx xxxxxx 5 xxxxxx xxxxxx 7 xxxxxx xxxxxx 9 xxxxxx xxxxxx 11 xxxxxx xxxxxx ANSWERS TO QUESTIONS Answers have been removed from the online versions of Vernier curriculum material in order to prevent inappropriate student use. Data tables have also been obscured. Full answers and sample data are available upon request by contacting info@vernier.com.