DNA Gel Electrophoresis Grade Level: High School, 9-12 and Advanced Placement Biology Objective The purpose of today’s lab is to review the concept of gel electrophoresis and to gain hands-on experience with us ing DNA-based electrophoresis techniques. Background Gel electrophoresis is a widely used technique in research laboratories to separate DNA, RNA or protein. Negatively-charged nucleic acids or proteins are repelled by a negative electric current that is applied across the top of the gel. The gel is a matrix of cross-linked molecules that acts as a sieve through which nucleic acids can migrate. The speed at which nucleic acids move through the gel depends, in part, on the size and charge of those molecules. Large and slightly charged molecules move slowly through the gel while small and highly charged fragments travel quickly. Once electrophoresis is complete, the nucleic acids can be visualized with a dye such as ethidium bromide. What can gel electrophoresis tell us? Each individual (except for identical twins) has a unique set of blueprints or DNA. By separating the fragments, electrophoresis allows us to diagnose genetic diseases, solve crimes, and determine paternity. Materials Electrophoresis Chamber (See http://gslc.genetics.utah.edu to construct your own apparatus) Agarose Water Erlenmeyer flask Graduate cylinder Aluminum foil or plastic wrap Balance Hot plate or Microwave Paper towels Masking Tape Electrophoresis Buffer Weigh boat or wax paper Latex Gloves Goggles Procedure Making the gel: How much volume can your gel form hold? __________ (To measure the volume, multiply the width, height, and length of the gel form. When you measure the height, take into consideration where the agarose should come up to on the comb used to create the wells.) You will make a 1% agarose gel. Given the volume of the gel form, how much agarose will you need? __________ (For example, if the volume of the gel form is 100 ml then 1 gram of agarose is needed to make a 1% gel.) Weigh out the amount of agarose on wax paper and carefully pour it into an Erlenmeyer flask that is 2-3 times the volume of the gel form. Prepare the gel form by taping the ends with masking tape (see Figure 1) or by using the rubber sealers (if provided). Be careful that the gel form is sealed well; otherwise, the agarose solution will leak onto your work surface before it has a chance to solidify. Measure the volume of TBE buffer to dissolve the agarose into a 1% solution. (Note: If you are using EDVOTEK’s 50X electrophoresis buffer, dilute the solution in the following manner: 6.25 ml of 50X buffer with 307.75 ml of distilled water to yield a total volume of 312 ml of dilute buffer. Use this buffer to fill the electrophoresis chamber in Step 1 of Loading the Gel. You may need to make more of the 1X TBE buffer.) Place the contents of the flask on a hotplate. Gently and constantly swirl the contents until the agarose is completely dissolved. You can check to see if the agarose is in solution by holding the flask up to the light and looking for floating particles. Your solution should be clear. Allow the solution to cool to 50-55ºC or until you can place your hand on the flask. If your solution begins to solidify before you have a chance to pour it, reheat the solution on the hotplate. Place the comb in the gel form. Pour the agarose solution into the gel form to where is comes a little over halfway up the comb. Allow the solution to set (solidify) undisturbed. It should look like milky jello. Preparing the samples Make the loading buffer as instructed on attached sheet. Why is glycerol used in the loading buffer? _______________________________________________________ Mix your samples with the loading buffer. Loading the gel Remove the comb and place the gel form into the electrophoresis chamber filled with 1X TBE buffer. The buffer should cover the gel. Note the lane that each sample is placed in. Use a clean micropipet tip or Pasteur pipet to load each sample. Using both hands to steady the pipet tip, position the tip over the well and gently expel the sample into the well. Too much pressure will flush the sample out of the well and into the buffer. Be careful not to puncture the bottom of the gel; otherwise, your sample will leak out of the bottom. Running the gel Once all the samples have been loaded into the wells, place the cover over the electrophoresis chamber. Is your gel oriented in the right direction:? See attached figure. Attach the electrodes to the electrophoresis chamber and then to the power supply. Place the cover over the electrophoresis apparatus. Your teacher will turn on the power box. Do not stick your fingers or anything else into the buffer. You will get electrocuted! Run the gel at 50-100 volts for approximately 60-95 minutes. When the DNA fragments have been separated, turn off the power supply. Unplug all the electrodes. With gloved hands, remove the gel form from the electrophoresis chamber to stain the gel. Staining and visualizing the gel Make a dilute solution of Methylene Blue Plus ™ Staining Solution (EDVOTEK, West Bethesda, MD) by mixing 60 ml of concentrated staining solution with 540 ml of distilled water. Take the gel and place into a rectangular plastic container with gloved hands. Remember, remove the gel from the electrophoresis chamber. Staining the gel in the chamber will ruin the electrophoresis apparatus. Pour the diluted staining solution over the gel until the gel is completely submersed. Stain the gel for 20-30 minutes. Your gel should appear as a dark blue piece of jello. Prior to de-staining, pour the staining solution into a glass bottle and store at room temperature. You can reuse this solution for up to 5 times. To de-stain the gel, submerse the gel in distilled water. You may need to pour off the water and add fresh water to the container as the stain leaches out from the gel. If the gel is too dark, de-stain overnight. If the gel is too light, repeat the staining process. Note: Agarose gels can be sealed in plastic wrap and stored in the refrigerator for several weeks. When gels are no longer needed, dispose of them in the trash. Clean-up Wash all equipment with tap water to remove any residual reagents. Avoid any detergents.