Introduction: Building and Running a Homemade Agarose Gel Electrophoresis
Introduction
A gel electrophoresis is a tool utilized by molecular geneticists to separate and view different parts of macromolecules such as DNA, RNA, or proteins. This technique works because most macromolecules are negatively charged. When they are placed in the gel wells on the negative end of the chamber and an electric field is applied, the molecules will migrate to the positive end through the gel’s microscopic holes. The smaller and more negative the molecules are, the further they will migrate to the positive end. This results in separation of the macromolecules based on mass and charge. If the molecules are dyed, a person can observe the distinct bands and interpret the results. The results of a gel electrophoresis have many applications, including forensic investigations, paternity tests, and measuring protein levels.
This Instructable explains all the steps necessary to gather the necessary materials and tools, construct your own gel chamber and comb, make a 1% buffer solution, make a1% Agarose gel, and run the gel electrophoresis with different colors of food dye. This will allow you to separate and compare the molecules in three colors of food dye.
This experiment is recommend for people of at least high school age wanting to learn about the principles behind this molecular genetics technique through actively experimenting. Knowledge of molecular genetics or the associated lab processes are not required to conduct this experiment.
The end of this document also contains a section to aid you in interpreting your results. A troubleshooting section is also provided to help you have a successful experiment.
Safety Hazards
None of the materials required for this experiment present any significant chemical or health hazard. While the Agarose powder and/ or gel is not toxic, be sure NOT to ingest it. Also, show caution when setting up and running the battery-powered electric current. Care should also be taken when handling the heated Agarose gel. Be sure to wear oven mitts when handling the heated Agarose gel.
Step 1: Materials/Equipment List and Cost/Time
Materials/Equipment List
Materials
Small, rectangular plastic box –A travel soap box will work well.
Stainless Steel Wire – 20 Gauge A Piece of Foam or Cardboard – At least 4 by 4 inches.
5– 9 Volt Batteries
Agar Powder – 1/4 teaspoon (1 gram)
Baking Soda– 1/2 teaspoon (2 grams)
Distilled Water– 200mL
2 Alligator Clips with Leads
Food Coloring Dyes – Red, Blue, and Green
Tools
Wire Cutters
Scissors
Measuring Spoons – Size: 1/4 and 1/2 teaspoons
Small Mixing Bowl
Microwave-Safe Bowl
Heat-Resistant Spoon
Microwave
Plastic Syringe (mL)
Butter Knife Ruler (cm)
Cost and Time
One run of this experiment may cost you between $50 to $75. This price range is subject to vary depending on how many items you do not currently posses. This prices range does not include common house hold items: microwaves, bowls, oven mitts, rulers, scissors, wire cutters, pencils, butter knifes, and spoons. This experiment should take approximately 1 hour and 30 minutes.
Purchasing Notes
All of these materials and tools can either be found at Walmart and/or Amazon.com. If you are wishing to run the experiment multiple times, you will need to purchase more batteries, since they will be drained fairly quickly. Five fresh batteries are needed each time the experiment is ran. Some sellers of Agar Powder could require you to be associated with a school.
Step 2: Procedure: Part 1: Gel Electrophoresis Chamber Preparation: Step 1
1. With your wire cutters, cut out two equal pieces of the stainless steel wire.
Both of these pieces should be about 2 cm longer than the width of your rectangular, plastic box.
This box will serve as the gel chamber. The wires need to be longer than the width of the gel chamber so they can still span across its width after the next step has been completed.
Step 3: Procedure: Part 1: Gel Electrophoresis Chamber Preparation: Step 2
2. Bend the extra 2 cm of wire into a hook.
This will allow the wire to hook over the side of the chamber. Please see the Figure #2 as an example.
Step 4: Procedure: Part 1: Gel Electrophoresis Chamber Preparation: Step 3
3. Place one wire on one end of the chamber, and the second wire at the other end.
Please see Figure #3 as an example.These wires will serves as the positive and negative electrodes, respectively.
Step 5: Procedure: Part 1: Gel Electrophoresis Chamber Preparation: Step 4
4. Assemble the 5 9-Volt Batteries.
Please see Figure #4 as a guide on how to assemble the batteries. A positive terminal of one battery will be snapped into a negative terminal of another battery. You need to continue this process with all the batteries until one positive and one negative terminal is not snapped to another battery.
Step 6: Procedure: Part 1: Gel Electrophoresis Chamber Preparation: Step 5
5. With your pencil, trace out the gel comb from either craft foam or cardboard. Then cut it out with scissors. Please see Figure #5 as an example of tracing the comb.
Please use Figure #6 as a reference to how the comb should look.
The comb will need to be wider at the top so it can rest on the edges of the chamber. Slits can be cut into the outer edges of the comb. This will allow it to stay upright easier.
The bottom of the comb needs to have three teeth for each color of food dye.
Each tooth should not be touching the bottom of the gel chamber. There needs to be around 1/2 cm of clearance between the bottom of the teeth and the chamber.
The teeth also need to be evenly spaced away from each other. Please see Figure #7 as an example.
The size and spacing of your teeth will depend on the dimensions of your chamber. A 10cm by 6 1/2 cm chamber will require at least 1 cm spacing for 3/4 cm teeth.
Step 7: Procedure: Part 2: 1% Buffer Solution Preparation: Steps 1 and 2
1. The buffer solution will need to be a 1% solution of baking soda. Combine 1/2 teaspoon of baking soda with 200 mL of distilled water in your mixing bowl. Use the plastic syringe to measure out the water.
2. Stir the solution well with a spoon.
Step 8: Procedure: Part 3: 1% Agarose Gel Preparation: Step 1 and 2
1. Place 1/4 teaspoon of agar powder in the microwave safe bowl.
2. Add 100 mL of the buffer solution made in the previous steps to the agar powder in the microwave safe bowl.
Step 9: Procedure: Part 3: 1% Agarose Gel Preparation: Steps 3, 4 and 5
3. In order to dissolve the agar powder, place the bowel in the microwave and turn it on for 1 minute. Do not forget to wear your oven mitts when handling the heated mixture. (Figure #10)
4. Every 15 seconds, stop the microwave and stir the solution.
Keep doing this process until the solution begins to bubble.
Once this happens, quickly and carefully remove the bowl from the microwave to avoid having the solution bubble over.
5. Make sure the solution is translucent. If it is not, you will need to remake your gel. Please reread through the steps to ensure you are following them correctly.
Please see Figure #11 in order to check the color of your gel.
Step 10: Procedure: Part 4: Preparing to Run the Gel Electrophoresis: Steps 1, 2, and 3
1. Make sure you have made the gel chamber, comb, buffer solution, and agar gel before moving on to this set of instructions. (Figure #12)
2. Next with the metal electrodes out of the plastic gel chamber, place the comb into the gel chamber.
3. The comb should be about ½ cm from one of the ends of the gel chamber. This 1/2 cm will provide room for you to cut a line in the gel in a later step with a butter knife. (Figure #13)
Step 11: Procedure: Part 4: Preparing to Run the Gel Electrophoresis: Steps 4 and 5
4. Now it is time to carefully and slowly pour the 1% Agarose gel into the gel chamber. Stop pouring when the Agarose gel has covered approximately 1/2 cm of the comb’s teeth. (Figure #14)
Tip: You can utilize the plastic syringe to move the Agarose gel into the chamber. Be sure to rinse the syringe out with distilled water after you have finished this.
Depending on the size of your chamber, you may not need to pour all of the Agarose gel.
5. Place the gel chamber in a safe place and wait at least 30 minutes for the Agarose gel to solidify. The gel will have the consistency of jello when it has reached room temperature and is ready. Do NOT proceed on to the following steps until you are sure the gel has completely cooled!
Step 12: Procedure: Part 4: Preparing to Run the Gel Electrophoresis: Step 6
6. Gently pour the rest of the buffer solution over the top of the gel in the chamber. The buffer solution should completely cover and submerge the gel. (Figure #15)
Depending on the size of your chamber, you may not need to pour all of the remaining buffer solution.
Step 13: Procedure: Part 4: Preparing to Run the Gel Electrophoresis: Step 7
7. Next, firmly grab onto the top of the gel comb and carefully pull it straight up and out of the gel. (Figure # 16)
Be extra careful with this step. The wells formed by the comb in the gel will hold the food coloring for this experiment.
Step 14: Procedure: Part 4: Preparing to Run the Gel Electrophoresis: Steps 8 and 9
8. You will need to make room for the wire electrodes. Take a butter knife and cut 2 lines across the width of the chamber on both ends. You can cut all the way down to the bottom of the chamber.
Please see Figure #17 below as a guide.
9.Then, place the wire electrodes back in the chamber. The electrodes should have the same placement as before. Refer back to Figure #3 above for a reminder of the electrode placement.
It is highly important the entire length of the electrodes are under the surface of the buffer solution. The hook of the electrode does not have to be submerged though.
Step 15: Procedure: Part 5: Running the Gel Electrophoresis: Steps 1, 2, 3, 4, 5 and 6
1. One at a time, each of the three different colors of food coloring will need to be placed in its own well with the syringe. Only a couple drops of each dye are needed
The less dye placed in the well, the clearer the results of the experiment will be in the end.
Tip: Place the tip of the syringe loaded with food coloring beneath the surface of the buffer solution and slightly inside the well before you push the plunger to release the dye.
Figure # 20 above depicts the technique of loading food coloring into a well.
2. First, fill the plastic syringe with red dye. Deposit the red dye into the well farthest away from you.
3. Rinse out the plastic syringe.
4. Next, fill the plastic syringe with blue dye. Deposit the blue dye into the middle well.
5. Rinse out the plastic syringe.
6. Finally, fill the plastic syringe with green dye. Deposit the green dye into the well closet to you.
Note: The color ordering in Figures #18 and #20 does not match up to the above schema.
Step 16: Procedure: Part 5: Running the Gel Electrophoresis: Steps 7, 8, 9
7. Place one clip of one of the alligator leads on the exposed positive battery terminal. Place the other end of this same lead onto the positive electrode. The positive electrode should be the wire farthest away from the wells.
8. Now, take the other alligator clip lead; one of its clips should be clipped onto the exposed negative battery terminal. Place the other end of this same lead onto the negative electrode. The negative electrode should be the wire closest to the wells.
9. At this point, you should be seeing bubbles bubbling up from the electrodes in the buffer solution because the current is passing through them.
If you do not see bubbles, please see the "Batteries" Section under “Troubleshooting” in the section below.
Please see Figure #18 and 19 for an example of these steps.
Step 17: Procedure: Part 5: Running the Gel Electrophoresis: Steps 10 and 11
10. The food coloring will migrate from the side of the chamber with the negative electrode to the end with the positive electrode.
11. Keep running the gel until food coloring bands have clearly and distinctly separated away from each other. While this process is occurring, be sure to check it at least every 5 to 10 minutes.
This whole process usually takes around 15 to 30 minutes.
Step 18: Interpreting the Results
The secondary color of food dye separated into different colored bands after the gel electrophoresis ran. This is because the secondary colored dye contained different sized macromolecules within it. Different sized molecules will separate at different speeds, resulting in bands at certain locations down the length of the gel. The same process occurs with DNA and other macromolecules.
Figures # 21 - 23 are sample results of this experiment.
Please note the order of the food dye colors are different from the previous instructions.
All the well lanes labeled "Green" have separated out into one band of yellow and one band of blue.
Step 19: Troubleshooting
Please read this section if you are encountering problems with this experiment.
Gel Comb
If you find that your comb’s material is not forming distinct enough wells, try using a sturdier material. One example is cardboard, if you are not already using it.
Agarose Gel
If your gel appears lumpy, most likely the Agarose powder did not fully dissolve. If you have not poured the buffer solution on it yet or ran the gel, you can place the gel back in the microwave safe bowl and re-melt the solution. Once the agar has fully dissolved, pour it back into the gel chamber to solidify. If you have already done one of the above tasks, you will need to start over with new agar and buffer solution.
If your gel is taking over 35 minutes to firm up, the ratio of agar to buffer solution is probably incorrect. You will need to remake the 1% Agarose gel with new materials.
Batteries
The most common problems with this experiment center around the batteries. If your gel is not bubbling when you attach the alligator clips, first check to make sure all the batteries are firmly and properly attached to one another. You can also check to see if the alligator leads are firmly attached to the batteries and the wire electrodes. If this does not solve your problem and you have used a battery before for a previous device or another gel run, please swap out that battery for a fresh one. Running the gel electrophoresis will really drain the batteries. If your solution is still not bubbling, you can try making new electrodes with fresh wire and using new alligator leads.
Dyes Keep Running Together?
Having a greater amount of space between wells will help keep the dyes from running together. This principle has already been implemented in your instructions. If you find that you are still having the dyes run together, try increasing the space between the gel wells by changing the dimensions of the teeth in the gel comb and rerunning the experiment. You might also try reducing the amount of dye you insert into the wells. It is okay if some of the dye comes up out of the well and slightly mixes with the other two colors nearby; however, try not to use too much dye.
Did Your Dye Not Run the Correct Way?
You probably got the positive and negative electrodes mixed up! Please review Figures #18 and 19 to ensure you are running the gel the correct way. Also make sure you have the alligator leads connected to the right battery terminal so they match up to their respective electrodes.