DNA Extraction Lab Honors Biology Purpose Students are interested in DNA. This allows students to extract DNA from several common materials. Below are the protocols for the extraction of DNA from organisms from 3 different kingdoms. The idea is to extract the DNA from each source, digest it with a restriction enzyme (Eco-R1 or HindIII) and then separate the fragments by gel electrophoresis and compare the results. Each extraction could be preformed by a different group and combined in one gel, so that each group of students can make a comparison. CALF THYMUS EXTRACTION Materials Calf Thymus (also called sweetbreads) may be cut into pieces and frozen for future use. Isotonic sucrose solution ( 106.1 grams table sugar in 1 liter of water ) Blender Cheese cloth Funnel Detergent solution ( 100 mL dishwashing liquid [ I've used Dawn], 15 grams NaCl [table salt works fine]: add distilled water to make a final volume of 1000 mL) ethanol (95%) dispensed into 3-5 mL amounts into screw top test tubes or small containers and stored in a freezer. One container per student group. 2 test tubes per student group inoculating loops or thin wires bent to form loop at the end for drawing up DNA from test tube As much as possible, you would like to work with cold materials and solutions. When the alcohol is removed from the freezer it should be carried on ice into the classroom. Procedure 1. MAKE A FEAR FACTOR SHAKE To break open the thymus cells: Add a small piece of thymus (slightly smaller than an egg) to the blender with enough sucrose solution to make a thin milkshake consistancy. Blend until thymus is broken up. Kids think this is cool and gross to watch! 2. Pour the blended thymus through a funnel which is lined with four thicknesses of cheese cloth. The filtrate collected below the funnel will contain many nuclei which can be observed under the microscope. This step removes gristle and other stuff. 3. Pour about 2 mL of the nuclei suspension collected from step 2 into a clean test tube for each student group. 4. Pour about 2 mL of detergent solution into another clean test tube for each group. 5. Students pour detergent solution into test tube of nuclei suspension and gently shake tube from side to side to mix. Do not shake enough to get foamy! In this step the detergent will break open the lipid membrane of the nuclei and release DNA. The salt in the solution will help neutralize the negative charges on the DNA molecules so they will precipitate. 6. Slowly pour COLD ethanol down the inside of the test tube containing the nuclei/detergent mixture so that a layer of ethanol forms on top of the mixture. DNA will come out of solution at the place where the two layers meet. 7. Use clean wood splints to dip into layer where the two solutions meet and stir gently. Students will be able to lift out masses of DNA. I have had students pull gently until they lifted about 2 meters of long chain molecules out. WHEAT GERM EXTRACTION Materials 250 ml beaker baking soda hot plate Adolph's natural meat tenderizer non-roasted wheat germ ice cold 95%#037; ethanol thermometer 15 ml test tube pH meter glass stirring rod Palmolive detergent distilled water test tube rack or 250 ml beaker graduated cylinders (10ml and 100ml) Solutions Baking soda solution: Add baking soda to distilled water until a pH of approximately 8.0 is reached. Protocol 1. Add 100 ml distilled water to a beaker and heat to 50-60oC. 2. Add 1.5 g wheat germ and mix until dissolved. 3. Add 5 ml detergent. Maintain 50-60oC temperature and stir for 5 minutes. 4. Add 3 g meat tenderizer. 5. Add baking soda solution to bring the pH to approximately 8.0. 6. Maintain the 50-60oC temperature and stir for 10 minutes. 7. Remove from heat. 8. Add 6 ml of the solution to a test tube and cool to room temperature. 9. Pour 6 ml ice cold ethanol carefully down the side of the tube to form a layer. 10. Let the mixture sit undisturbed 2-3 minutes until bubbling stops. 11. The DNA will float in the alcohol. Swirl a glass stirring rod at the interface of the two layers to see the small threads of DNA. YEAST EXTRACTION Materials dry yeast Palmolive detergent Adolph's natural meat tenderizer graduated cylinders (10ml and 100ml) beaker blender distilled water 15 ml test tube non-iodized salt ice cold 95&037; ethanol glass stirring rod test tube rack or 250 ml beaker Solutions detergent/salt solution: 20 ml detergent 20 g non-iodized salt 180 ml distilled water 5% meat tenderizer solution: 5 g tenderizer 95 ml distilled water Protocol 1. Mix 1 package of dry yeast with 40 ml of 50oC hot tap water to dissolve the yeast in a beaker. Keep mixture covered and warm for about 20 minutes. 2. Add 40 ml detergent/salt solution. 3. Place mixture in a blender and blend 30 sec-1 minute on high. 4. Pour mixture back into the beaker, add 15 ml of meat tenderizer solution, and stir to mix. 5. Place 6 ml of mixture into a test tube. 6. Pour 6 ml of ice cold ethanol carefully down the side of the tube to form a layer. 7. Let the mixture sit undisturbed 2-3 minutes until bubbling stops. 8. You will see a precipitate in the alcohol. Swirl a glass stirring rod at the interface of the two layers. The precipitate is DNA. Note: There restriction enzyme digest protocol is in the process of being developed. Feedback as to what is working and what needs to be modified is welcome. RESTRICTION ENZYME DIGEST After each extraction, add a restriction enzyme (Eco-R1 or Hind III) 1. To a very small amount of DNA, 6L of distilled water, 1L buffer, and .5L restriction enzyme 2. Restriction enzymes and buffers must be stored in the freezer 3. Tap the tube to mix the contents 4. Incubate 2 hrs. to overnight 5. After incubation, place in freezer or place into aragarose gel for electrophoresis GEL ELECTROPHORESIS 1. 2. 3. 4. 5. 6. Put 6 L of digest into the well of a pre-made agarose gel Place gel into electrophoresis chamber and cover in buffer Run gels until last band is near end of gel. Stain for 20-30 minutes Destain overnight Observe bands Possible questions to ask the student: 1. What is the consistency of the DNA? 2. Is this what you expected? Explain 3. Explain what was happening in each step. That is, why was each step necessary for getting the DNA out of the cells a. Blending b. Filtering c. Detergent 4. What was the function of the alcohol? 5. Compare and contrast the results in the banding patterns of the DNA from the 3 sources. 6. Explain why there were differences in the different DNAs 7. Why were there multiple bands in the gel for each DNA source? 8. If What affects the ability of DNA to move through the gel. 9. we were able to extract DNA from your tissue predict what it may look like. 10. Relate your prediction to evolution.
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