Exercise 1 Extraction of DNA In this exercise, you will extract and observe DNA from split peas. Note: The night before beginning the experiment, place the 91% isopropyl alcohol into a freezer to chill. The entire bottle may be placed in the freezer without harming the bottle or its contents. If a freezer is not available, place alcohol bottle in an ice-bath at least 3 hours before beginning the experiment. Note: If a blender is not available, a mortar and pestie may be used and the split peas must be soaked overnight in distilled water. Procedure 1 Break down the pea tissues with a blender or by hand with a mortar and pestie. Use 1 of the following 2 methods: Blender Method • Retrieve the split peas and 2 salt packets from the kit. • Add the split peas and both salt packets into the blender. • Measure 1 cup of cold water and add it to the blender. . Place the lid onto the blender and blend the split peas, water, and salt mixture on the high setting for approximately 30 seconds. Mortar and Pestie Method • Retrieve the split peas from the kit • Measure 1 cup of distilled water and pour it into 1 of the drinking glasses, • Add the split peas to the drinking glass with the distilled water and use the spoon to mix the peas with the distilled water • Allow the split peas to soak overnight in the distilled water • After the split peas have soaked overnight, use the spoon to transfer them into the mortar. Try to transfer as little of the distilled water into the mortar as possible • Gather a second clean drinking glass and add cup of distilled water and the 2 salt packets. • Use the stirring rod to fully mix the salt into the water until it is completely dissolved. • Use the spoon to transfer the salty water into the mortar until it is full. See Figure 5. Xu Figure 5. Mortar is full with peas and salty water. • Pick up the pestle and use it to break open and crush the peas. • Continue to crush the peas with the pestie until they are fully incorporated into the salty water. 2 Gather a clean drinking glass and put on your safety gloves and glasses. 3 Remove the cheesecloth from the kit, place it over the drinking glass, and secure the cloth into place with the rubber band. 4 Gently pour the contents of the blender or mortar into the cup, allowing it to strain through the cheesecloth Note: The solution in the cup, after straining, contains the pea DNA. 5 Carefully remove the rubber band and cheesecloth from the drinking glass. Set the glass holding the DNA solution aside. Discard the cheesecloth and the solids in a trash bin. 6 Blender Method: To the strained fluid remaining in the drinking glass, add the entire dropper full of dish soap and carefully stir the solution with the stirring rod. Mortar and Pestie Method: To the strained fluid remaining in the drinking glass, add the dropper full of dish soap and carefully stir the solution with the stirring rod. 7 Rinse the stirring rod with water. 8 Place the solution into the refrigerator or on ice for 10 minutes 9 After 10 minutes, gently pour the cold soap/DNA solution into the 25 x 150 mm test the vintill the whole 9 After 10 minutes, gently pour the cold soap/DNA solution into the 25 x 150 mm test tube until the tube is 1/3 full 10 Gather the meat tenderizer (papain powder) from the kit. 11 Carefully transfer approximately the papain powder into the test tube by tapping the papain powder bottle over the test tube. 12 Use the stirring rod to gently stir the papain powder into the solution, so as not to break up the DNA. 13 Remove the 91% isopropyl alcohol from the freezer or ice bath. 14 Hold the test tube at a 45° angle and slowly add ice-cold isopropyl alcohol from the bottle until there are equal amounts of alcohol and solution 15 Gently move the test tube back to an upright position and allow the contents to settle for 1 minute. 16 Observe the interface of the 2 layers; DNA will start to precipitate as white, stringy filaments 17 Observe the DNA that was extracted from the split peas. What does it look like? Is there more or less DNA than you expected to see? The answers to these questions should be included in your report. 18 When you have finished the experiment, pour the contents of the test tube down the sink and rinse with water. 1. Describe the physical appearance of the pea DNA. B ETT, O Words 2. Were your experimental results what you expected to see? Specifically, was there more or less DNA than you expected? в то 13 вт т, O Words 3. What was the purpose of adding the detergent to the experiment? If the detergent was not used, do you think the experiment results would have changed? Explain your answer. в то 18 вт т. O Words 4. If you used 5% isopropyl alcohol instead of 91%, do you think the experiment results would have changed? Explain your reasoning.