Name: _______________________________ DNA Extraction Laboratory Introduction: DNA is found in the vast majority of an organism’s cells. This molecule functions in providing the plans for building the cell’s proteins. Proteins are vital to the cell, functioning as structural components as well and functional molecules of the cell. Enzymes and hormones, for example, are primarily proteins. DNA is located in three different structures within eukaryotic cells. Nuclear DNA is located in the nucleus; mitochondrial DNA is located in mitochondria, and; chloroplast DNA is located in the chloroplast of eukaryotic photosynthetic cells. By far, the greatest amount of DNA is located in the nucleus of eukaryotic cells. In this laboratory investigation, you will attempt to extract DNA from two different organisms using slightly different methods. Make sure you following the directions exactly in order to ensure success. Method: Although the methods used for DNA extractions vary somewhat, the goals remain the same: release nuclear DNA from the cell’s nucleus, release the nuclear DNA from the cell, and to precipitate DNA out of solution so that it can be seen. Common components of a DNA extraction lab include: Detergent Detergent contains sodium laurel sulfate, which cleans dishes by removing fats and proteins. It acts the same way in the DNA extraction protocol, pulling apart the lipids and proteins that make up the membranes surrounding the cell and nucleus. Once these membranes are broken apart, the DNA is released from the cell. Salt Because each nucleotide of the DNA molecule possesses a negatively charged phosphate group, DNA is a negatively charged molecule. Therefore, DNA molecules are not attracted to each other but are repelled by the like charges they possess. Adding salt (NaCl) to a solution containing DNA neutralizes the negative charges of the DNA molecule, making the separate DNA molecules more likely to collect together and become visible. Salt is able to do this because the sodium and chloride ions separate in solution and the positive sodium ions (Na+) are attracted to the negative charges of the DNA’s phosphate groups, thus neutralizing the negative charge. Alcohol The DNA released from the cell nucleus is dissolved in the water/detergent/specimen solution and cannot be seen. DNA precipitates out of solution in alcohol, where it can be seen. In other words, DNA is less soluble in alcohol than in water. When alcohol is added, the separate DNA molecules clump together and become visible. In this activity, you will extract DNA from two different organisms: A strawberry, and a human (you!) Strawberries are octoploid—meaning they have 8 copies of each chromosome—so there is plenty of DNA to be found in a strawberry. By contrast, humans are diploid—two copies of each chromosome. What you will need: A strawberry (a 1” chunk of banana or kiwi will also work) Deionized water (bottled water or tap water that has been sitting in an open cup for 6-12 hours) Salt Clear dish detergent Rubbing alcohol (70 percent solution or higher) Blue food coloring A funnel A coffee filter or cheesecloth Several clear glass or plastic cups or jars A Zip-close baggie Part 1: Strawberry DNA extraction: DNA Extraction Buffer (can be prepared in advance): 900 ml of deonized water, 50 ml of clear dishwashing detergent, 2 teaspoons of salt 1.Add a strawberry (or half) to a Ziploc sandwich bag and use your hands to smash the strawberry, taking care to not break the bag. 2. Add 10 ml of the DNA extraction buffer (recipe above). (about 1Tbs+1 tsp) 3. Smush the strawberry and buffer for about one minute. 4. Use a funnel and coffee filters to filter the strawberry juice into a single test tube, beaker, or glass jar 5. Slowly pour cold ethanol or 91% isopropyl alcohol over the top of the strawberry mixture. It may be best to use a pipette to add it. You want a single layer of clear ethanol on top of the strawberry mixture. COLD ethanol will work best. 6. White strands will form in the ethanol layer, use a stirring rod to spool the strands. Describe the DNA you have extracted. PART 2: Human DNA Extraction For a short video tutorial on extracting your own DNA, see http://www.planetscience.com/categories/experiments/biology/2012/03/extract-your-own-dna.aspx The video also contains some further information that can help you answer the questions at the end. 1. Mix 1 cup bottled/deionized water with 1 Tbs salt in a cup. Stir until salt is dissolved. 2. Transfer 3 tbsp of the salt water into a separate cup. 3. Gargle the salt water for 1 minute. Don't swallow it! 4. Spit the water back into the cup. Your cheek cells are now in the salt water. 5. Add one drop of washing up liquid to the salt water. Stir gently. Try not to create any bubbles. 6. In a separate cup, mix 100 ml isopropyl alcohol and 3 drops food coloring. 7. Gently pour the alcohol and food coloring mixture into the salt water cup. Tilt the salt water cup as you pour, so the alcohol mixture forms a layer on top of the salt water. Wait for 2.5 minutes. You should see white clumps and strings forming. This is your DNA. Describe the DNA you have extracted. FOLLOW UP QUESTIONS: 1. Note the similarities and differences between the two samples. What might account for the differences? 2. Describe the role of the detergent in DNA extraction. 3. Describe the role of the salt solution in DNA extraction. 4. Describe the role of alcohol in DNA extraction. 5. What are some reasons why scientists might want to extract DNA?
