Separation of a Mixture using Gel Electrophoresis

Background:
As a junior chemist working under contract with the EPA, you are preparing to embark on your first assignment. It has been leaked to the agency that a small manufacturer of prepared foods has been dumping waste products into a stream feeding into the upper Shenandoah. The FDA has been tipped that the small company may be illegally using a banned substance, red dye #34, shown to be carcinogenic in rats, as a food additive. FAD has never discovered it during its inspections, however, and they are asking for EPA's help in checking on a spill of dark-colored substances near the company's plant.

Assignment:
You and two colleagues are to collect samples near the site of the spill and provide a quick analysis in order to alert FDA for an unannounced visit. Can you design an analytical procedure using gel electrophoresis, which could be carried out in a mobile lab near the site and is both fast and reliable? You need to plan on running multiple samples of the effluent from the plant taken over several days. The mixture of food dyes you anticipate analyzing should be separated enough on agarose gels to cleanly identify the bands without sacrificing too much time or gel materials.

Your objective in designing the analytical procedure is to come up with an effective separation of dye substances, which takes only 15-25 minutes per run. To do this you should examine several concentrations of agarose (effecting pore size) for ban separation over various time periods.

Objective:
To practice using the micropipette ad other lab tools.
To use the process of gel electrophoresis to separate mixture.
To identify the chemicals in the water sample obtained from the river.

How Gel Electrophoresis Separate Sample Mixture

The sample is mixture of several chemicals which all have their characteristics colors in certain pH environments. These molecules carry a certain numbers if positive charges or negative charges in their ionic form, and as a result they travel in different speeds at a specific voltage.

Specific gel electrophoresis uses electricity to pull the sample mixture through the gel. As the sample travels through the gel, tiny holes in the gel trap the large sample particles. In this way, smaller particles will travel farther and separate from the mixture. Each sample mixture creates a unique pattern depending on the size of the particles.

The flow pattern of each sample will also be affected by type of charges the sample particles carry. Notice that the gel electrophoresis box has a red and black end: The red has a positive charge and black end has a negative charge. Since opposite attract, the positive end of the box will attract sample particles with a net negative charges (Negative Ions). Similarly, the negative end of the box will attract particles with a net positive charge (Positive Ions).

Procedures:

DAY 1. Preparation of Buffer solution.

1. Obtain a 250ml of reagent bottle.
2. Add about 5ml of 10 x buffer stock solution into the bottle, and then test the pH value of the solution with pH meter.
3. Add 1M NaOH drop by drop and test the pH value until the pH is about 7.5 - 8.5. If the original buffer pH is below 7, add 1M HCL drop by drop till the pH value of the solution reaches 7.5 - 8.5. Record the exact pH value of buffer solution in the data table.
4. Add about 130ml of deionized water to dilute the buffer to 1X concentration.
5. Label the bottle with your group name and save the solution for the next day's experiment.

Cast Agarose Gel
1. Seal ends of the gel-casing tray with tape, and insert well-forming comb. Place gel- casting tray out of the way on the lab bench so that the agarose poured in the next steps can set undisturbed. Repeat the above to make second tray.

Use Erlenmeyer flask to prepare one of the following reagent of gels, 0.8%, 1.2% and 2.0%

2. Measure either 1.2 g agarose (for 0.8%), or 1.8g (for1.2%), or 3.0 g (for 2.0%) on the electronic balance then transfer the agarose into the Erlenmeyer flask.
3. Add 150 mL of 1 X buffer solution you made to the flask.
4. Heat the agarose solution on the hot place and stir the solution constantly until the agarose is totally dissolved.
5. Carefully pour enough agarose solution into casting tray to fill to depth of about 5 mm. Gel should cover only about one-third the height of comb teeth. Use a pipette tip to move large bubbles or solid debris to sides or end of tray, while gel is still liquid.
6. Gel will become cloudy as it solidifies (about 10 minutes). Be careful not to move or jar casting tray while agarose is solidifying. Touch corner of agarose away from comb to test whether gel has solidified.
7. Label a plastic zippered bag with your group's name.
8. When agarose has set, unseal ends of casting tray, place the gel in a plastic tray and seal in the zippered bag. Place the bag in the proper place.

Day 2. Separation of sample in a fixed time period

1. Place the gel on the platform of the gel box so that comb is at the negative (black) electrode.
2. Fill the box with TBE buffer to a level that just covers entire surface of gel.
3. Gently remove comb, taking care not to rip wells.
4. Make certain that sample wells left by comb are completely submerged. If "dimples" are noticed around wells, slowly add buffer until they disappear.
5. Use micropipette to load 10 uL of the sample or standard into the separate well in the gel. Use fresh tip for each reaction.
a. Steady pipette over well using two hands.
b. If there is air in the end of the tip, carefully depress plunger to push the sample to the end of tip. (If air bubble forms "cap" over well, loading dye will flow into buffer around edges of well.)
6. Close top of electrophoresis box, and connect electrical lid to a power supply, anode to anode (red-red) and cathode to cathode (black-black). Make sure both electrodes are connected to same channel of power supply.
7. Turn power supply on, and set to 120 volts. The ammeter should register approximately 50-100 milliamperes. If current is not detected, check connections and try again.
8. Electrophoresis for 15 minutes. Good separation should have occurred in that time.
                                                      STOP HERE NOW
9. Turn off the power supply, disconnect leads from the inputs, and remove top of electrophoresis box.
10. Carefully remove casting tray from electrophoresis box, and slide gel onto a piece of paper and measure the length of each band and record on the data table 1.
11. Place the gel back to the casting tray and then place the casting tray back into the electrophoresis box.
12. Repeat procedures 6 and 7.
13. Electrophoresis until the bands approach one centimeter away from the end of gel.
14. Turn off power supply, disconnect leads from the inputs, and remove top of electrophoresis box.
15. Carefully remove casting tray from electrophoresis box, and slide gel onto a piece of paper then measure the length of each band and record them into the datable 2.

DATA TABLES AND FOLLOW-UP QUESTIONS

Data Table 1.
Percent of gel____________
pH of your buffer solution______________

	Sample 1	Sample 2	Sample 3	Unknown
Time
Length
Color

Data Table 2.
Percent of gel __________
pH of your buffer solution ___________

	Sample 1	Sample 2	Sample 3	Unknown
Time
Length
Color

Follow up Questions:

1. List your known sample in terms of sample 1, sample 2, or sample 3.
2. How far has the mixture traveled? Does mixture separate into different colors?
3. Is the mixture moving toward the positive end or negative end?
4. Does time of run affect the separation result of the mixture? What may occur if the running time is too short or too long? Explain.
5. Does the gel concentration affect the separation of the mixture?
6. Compare your data to the data from other groups. At the same time of run, which concentration of the gel shows the optimum separation of the mixture?
7. Carefully considering both gel running time and the cost of gel material, evaluate the entire procedure. What concentration of agarose and the time of run will result in the optimum separation you need? Explain your conclusion.