NAME: ________________________________ SEPARATING DNA MOLECULES BY AGAROSE GEL ELECTROPHORESIS PURPOSE To perform agarose gel electrophoresis on DNA samples. To understand the process of gel electrophoresis and how it separates DNA fragments based on size. INTRODUCTION DNA molecules can be analyzed using agarose gel electrophoresis. When an electric field is applied across an agarose gel, negatively charged DNA molecules will migrate through the gel matrix toward the positively charged cathode at a rate relative to the size of the DNA fragment. An analyst determines the size of DNA fragments by the migration distance; smaller fragments will move farther through the gel than larger fragments. Before the DNA samples can be loaded onto the gel they first need to be mixed with a loading buffer that contains glycerol which helps the samples remain in the wells and tracking dyes. When current is applied, not only does the DNA move but so do the tracking dyes. Within five to ten minutes two or more distinct bands of dye appear. These are markers that show the progress of DNA migration in the gel, since the DNA can not be seen at this point. After the gel has run to completion, it is then stained with a dye that adheres to the DNA due to its negative charge, and does not bind tightly to the gel matrix. The gel is often put in a destaining solution to remove excess dye that may nonspecifically bind to the gel matrix. When the destaining process is complete it is now time to view the gel. Distinct bands formed by DNA molecules of the same size appear when the gel is transilluminated on a light box. At this point the DNA bands can be analyzed to determine the distance migrated relative to DNA bands of known size. Now let’s try to run a gel! MATERIALS Latex Gloves Safety Glasses Loading Buffer Gel Box Microtube Rack Microtubes Nucleostain 1 Tube Crime Scene DNA 4 Tubes of Suspect DNA 0.5-10 uL Micropipetter 0.5-10 μL Micropipetter Tips Container for Tip Disposal DNA Ladder 1 X TAE Buffer PROCEDURE 1. Before beginning be sure to glove your hands and wear safety glasses. 2. Have a properly prepared gel box as described in “Preparing and Agarose Gel”. The gel should be polymerized, oriented such that the wells are near the negative pole (black) and flooded and submerged with 1X TAE buffer. 3. In a microcentrifuge tube rack place: Sample DNA Microcentrifuge tubes containing suspect DNA Microcentrifuge tubes containing crime scene DNA Microcentrifuge tube containing DNA ladder Microcentrifuge tube containing loading buffer 6 empty microcentrifuge tubes 4. Using a permanent marker label 5 microcentrifuge tubes as follows: suspect A suspect B suspect C suspect D crime scene Label the microcentrifuge tube containing the DNA ladder as well. 5. Using a 0.5-10 uL micropipetter place 5 uL of loading buffer into each of the five empty tubes marked for sample DNA. In the empty microcentrifuge tube that will contain the DNA ladder add only 3 uL of loading buffer. To save time and tips it is not required to change tips between tubes at this point. 6. Using a fresh tip, transfer 10 uL of suspect A DNA into the corresponding microcentrifuge tube containing the loading buffer. Repeat this step with the remaining 4 suspect DNA samples, using a fresh tip each time. Be sure to change tips between each suspect sample. We don’t want to mix DNA! Helpful Hint: When adding the DNA to the loading buffer it is important to mix the two solutions together. This can be achieved by gentley pipetting the combined solution up and down 3 times. 7. Using a fresh tip add 3 uL of DNA ladder into the tube marked for the ladder. Be sure to mix the DNA with the loading buffer by using the above technique. Stop and read step 8 carefully! 8. Loading a well is a very delicate process. Follow the steps described below to ensure the wells are not damage during loading. Pull up the sample you wish to load using the micropipetter and begin loading by slowly and carefully placing the tip inside the well. Feel for the edges of the gel gently by moving the tip back and forth. Do not poke the pipette tip into the bottom or the sides of the gel. Slowly expel the sample into the well. Press the pipette button only to the first stop. There will be solution remaining in the tip. The second stop on a micropipetter expels a burst of air which serves to push the last drop of liquid out of a tip. This burst of air is strong enough to flush the sample out of the well. For a successful gel it is not important to expel this last drop of air. Slowly pull the pipette tip out of the well but do not release the plunger. This can cause the sample to be sucked back into the pipette tip. Once DNA samples have been loaded into the wells, be sure not to disturb or move the gel box. It is easy to flush the samples out of the wells. Be sure to change tips between each suspect sample. We don’t want to mix DNA! 9. Using a 0.5-10 uL micropipetter, load 6 uL of the ladder-dye mixture into the first well. Remember to change tips between each sample. 10. Load 10 uL of the DNA samples into the next 5 wells. Record the order in which these are placed into the wells. Remember to change tips between each sample. 11. Carefully place the lid on the gel box without disturbing or moving the box. Make sure that the color of the leads match (black to black, and red to red). Helpful Hint: Run the gel as soon as possible so that the sample does not diffuse out of the wells. 12. Plug the box into a power supply by matching the color of the leads (black to black, and red to red) in side by side sockets. 13. Set the Volts at 75 V, set the Amps to 185 A and set the time for 90 minutes. Make sure that the K button on the box is reading for Volts and not Amps. After checking the settings again, press the run button. Helpful Hint: If everything is working correctly little bubbles will rise from the thin wires running along the bottom of the gel box reservoirs. 14. After a few minutes check to see if the DNA sample is running from negative (black electrode) to positive (red electrode). Distinct blue and purple bands should appear within 5 to 10 minutes. This is the loading dye and can be used as a marker to follow the progression of the DNA. 15. When the gel is done running turn the power supply off, remove the electrodes from the power supply, and remove the lid from the gel box. 16. Gently take the gel tray out of the box and carefully slide the gel off into a plastic container. 17. Add Nucleostain solution until the gel is completely submerged. Place a lid over the container and let the gel soak overnight. 18. Once the gel has been allowed to stain overnight put on gloves and remove the gel from the staining container. Place the gel in an empty container and add 1 X TAE buffer until the gel is submerged. Helpful Hint: Nucleostain may be poured back into its original bottle and reused. 18. After a minimum of one hour, remove the gel from the destain solution and place it on the light box. 19. Photograph the gel and measure the distance the bands have traveled. QUESTIONS AND CONCLUSIONS 1. What special property of DNA allows for gel electrophoresis and staining adhesion? 2. Describe two precautions you take when preparing and running a gel? 3. What is the purpose of loading a ladder beside the samples?