How to Set Up and Run Gel Electrophoresis

Transcription

1 How to Set Up and Run Gel Electrophoresis

2 2 Introduction Gel electrophoresis is a process by which one can separate and analyze various macromolecules based on their size and charge. The process is most commonly used to evaluate DNA strands of varying sizes. In gel electrophoresis, an electric current is run through macromolecule samples of different sizes (base pairs). Because the samples are all charged, they will be attracted to one charge and will be move across the gel towards that electrode. The larger sized samples will not move as far as the lighter samples due to the larger mass. Therefore, you can compare the distance the samples travelled to a standard, or ladder, of a known length of base pairs. Gel electrophoresis is most commonly used to determine the length of and number of base pairs in a sample of DNA. Therefore, due to the negative charges in the DNA molecules, the samples will move toward the cathode (red lead). Tip: Remember the sample will move towards the cathode with the pneumonic device run to red. The materials required for gel electrophoresis are: Figure 1: Run to Red Chemicals 50X TAE solution (buffer) 1 L plastic bottle 250 ml flask 2.5 g agarose distilled water 1 µl Ethidium Bromide Loading buffer Casting materials Gel tray Casting gates Comb Microwave Electrophoresis equipment Pipette Pipette tips Standard/ladder Samples with dye Gel box and lid Power supply UV camera Microfuge tubes Safety Equipment Safety glasses Oven mitts Latex gloves Latex gloves Gel electrophoresis is a fairly common technique when working with DNA and should always be carried out in a laboratory setting. While running gel electrophoresis, you should use proper laboratory technique and observe all safety precautions outlined in this instruction set. Failure to do so may result in harm to yourself or others and may produce inaccurate results for the experiment. The procedure consists of three main steps: Preparing the Gel, Setting up the Components, Adding Samples and Running the Gel, and Viewing the Gel.

3 3 Preparing the gel 1. Pour 20 ml of 50X TAE solution into the 1L plastic bottle. 2. Fill the rest of the bottle with distilled water so the total volume is 1 L. This bottle now contains 1X TAE solution. 3. Using the 250 ml flask, measure out 250mL of 1X TAE solution. 4. Add the agarose to the buffer solution. 5. Put the mixture in the microwave for approximately 2 minutes, or until it boils. WARNING: Use oven mitts when taking the flask out of the microwave. The flask and solution will be very hot! 6. Take the flask out of the microwave and place on the counter to cool. While the agarose solution is cooling, set up the electrophoresis components. Setting up the components and Casting the Gel WARNING: Make sure to wear latex gloves and use caution when using ethidium bromide. It is considered to be a mutagen. Use the same precautions when touching the gel for the rest of the procedure because it now contains ethidium bromide. 1. Place the gel tray in the center of the gel box. 2. Place the gel casting gates in the slots in the gel box to section off the wells from the gel tray. 3. Place the comb with the tines pointing down (see Figure 2) in the comb slots of the gel tray, a little over ½ and inch away from the casting gate. 4. When the 250 ml flask containing the agarose solution is cool enough to touch with your bare hand, add 1 µl ethidium bromide to the solution and swirl the flask to mix it. 5. Pour the solution into the gel tray, making sure no solution leaks past the casting gates and into the wells 6. Leave the gel to cool for approximately 10 minutes. The gel should be firm, but not so firm that the comb is stuck in the gel. 7. Once the gel has cooled, carefully remove the casting gates and comb; making sure the gel retains its shape. Figure 2: Electrophoresis set up

4 4 8. Fill one gel box well with the previously prepared 1X TAE solution from the 1L plastic bottle. 9. Fill the other well with the same solution. Keep filling this well so the buffer overflows over the gel. 10. Continue pouring buffer solution until there is about 2-5mm liquid on top of the gel. Adding the samples and Running the Gel 1. Add 2 µl loading buffer to 8 µl of your first sample in a microfuge tube and swirl the tube to mix. 2. Repeat step 1 for each of your samples. 3. Using the micropipette, add 2µL of the ladder to the first well. Avoid using the wells closest to the outsides of the gel. Tip: When loading the samples, make sure to get the pipette deep enough to ensure the tip is in the well, but not too deep that it punctures the gel. Refer to Figure 4 for an idea of how the sample will sit in the well. After releasing the sample from the pipette, keep the plunger pushed down while removing the pipette tip from the buffer solution to avoid taking liquid up into the pipette. For more tips, refer to page 5 of this instruction set. 4. Follow step 3 for each of the samples. Tip: Make sure to make a note of which sample is in which well. 5. Place the gel box lid on the box and make sure it is on all the way and secure. 6. Using the leads from the power source, connect the red lead (cathode) to the box on the side farthest from the samples. 7. Connect the black lead (anode) to the box on the side closest to the samples. 8. Turn the power source on to about 90V for about minutes or until the dye has progressed about 75-80% towards the opposite side. There will be some bubbles formed by the electrodes. Note: The distance the dye has travelled does not represent the distance the samples have travelled. To view the progression of the samples, use a UV light. Figure 3: Pipetting

5 5 Pipetting Tips: The best way to keep the pipette steady is to rest your elbow on a solid surface and hold that hand stead with the other hand. If a well is punctured during pipetting, just avoid using that well and use the next one. If some sample has spilled out of the well and into the buffer solution, carefully use the pipette (with a fresh tip) to suck up the excess sample and move on to the next well. If the plunger is released while the pipette is still in the well, remove the pipette and put the liquid into a waste container. Add more buffer solution to replace that lost, and try again using the same well. Viewing the gel 1. Turn off the power source. 2. Remove the leads from the lid of the gel box. 3. While wearing gloves, carefully remove the gel tray and gel from the gel box and place on a paper towel. 4. Remove the gel from the gel tray and photograph using a UV camera. The samples will show up under UV light because of the addition of the ethidium bromide. The lines in the land with the standard ( L in Figure 5) can then be used to determine the lengths of the samples. Depending on the standard used, each line will correspond to a different length of base pairs. The samples that correspond to the lines from the standard contain DNA of the same number of base pairs. Figure 4: UV camera

6 6 Trouble Shooting and Final Tips Running gel electrophoresis can be a very tedious process. Make sure you have plenty of time and do not need to rush through the procedure. Use your time wisely by setting up and cleaning various components while other steps are taking place (such as cooling the agarose or running the gel). If the samples do not appear under UV light, you have forgotten the ethidium bromide. If there is no, or very little, separation between the samples, use a less diluted buffer. Finally, make sure to consult with other groups and data to avoid relying on distorted results and try to prevent the same mistakes from happening in the future. Gel electrophoresis can be an extremely helpful tool in macromolecule analysis, but it requires a steady hand, plenty of time, and an incredible amount of patience. Audience This instruction set is for college biology students in a laboratory setting. The students should already understand proper laboratory technique. They should understand the principles and theories behind the process of gel electrophoresis and have experience with micropipetting. The terms used in this instruction set are common in any biology laboratory. Resources Cited Title Page: content/uploads/2014/06/1984_pfgel1.jpeg power- of- dna- and- the- potential- for- misuse/ file:///users/christinefanning/desktop/base%20pairs Figure 1: file:///users/christinefanning/desktop/run%20to%20red Figure 2: file:///users/christinefanning/desktop/entire%20apparatus Figure 3: