DNA Isolation Lab DNA, or deoxyribonucleic acid, is the set of molecular instructions for building and running any organism. It is the genetic information to direct all cell activities. Remember, you are basically built of, and run, by proteins! DNA, for the most part, makes up the chromosomes of cells of all living things. It can be found in the nucleus of most living things. Watson and Crick were the first scientists to describe the structure of DNA as a double helix. OUR FUNDAMENTAL – or CLASS PURPOSE STATEMENT: This investigation is being conducted to see if DNA can be extracted from various sources as well as isolated so that it can be observed. 1. Create your problem statement in your journal. Perhaps the question we are asking is: What does DNA look like after it is extracted from _____ cells? Or, Does DNA extracted from _____ look the same as DNA from _____? Remember, the testable question is specific to the lab you are doing. You may need to look through the protocol before you can write a testable question. 2. Answer the following questions as your background section for the lab. Where in the cell is the DNA located? (Specifically) What does the term DNA stand for? What is the structure of DNA called (give 2 answers)? Why is DNA important? (What does it have in it that's important) What molecule does DNA "code" for (it is the directions to make what)? What chemical lysed (broke down) the membrane of the nucleus in your experiment? What chemical solution (s) protected the DNA in order to be able to extract it from cells? What chemical precipitates (transforms from liquid to solid) the DNA? 3. Identify the manipulated and responding variables. What variable are you and your partners changing? What are you ―measuring‖ as a result? Write a hypothesis in the form of an ―If…then…because‖ statement. 4. Create a Protocol section using the given materials and procedures list found on the next page. 5. Set up a data section in your journal (What kinds of data are you collecting?) Background Information: There are four basic steps of DNA extraction. a. The cell must be lysed (broken open) to release the nucleus. b. The nucleus (if present) must also be opened to release DNA. c. DNA must be protected from enzymes (special proteins) that will damage it. d. Once the DNA is released, it must then be transformed from liquid to a solid form. This is called precipitation and is done the use of cold alcohol. In order for the cell to be lysed, the lipid (fatty) molecules in the cell and nuclear membranes must be broken down. A detergent, salt solution and shaking accomplish this. Remember the commercial, ―Dawn takes grease out of the way.‖ Heat could be used. However, it would destroy the enzymes, as well as the DNA--that is why a detergent and a salt solution are used. The prepared solution uses Epsom salts and buffered aspirin to deactivate the enzymes that degrade (break apart) the DNA when released; the solutions also stabilize the DNA. Remember that DNA is supposed to stay in the nucleus. Enzymes will break down DNA if it is detected outside the nucleus. WHY DO YOU THINK THAT WOULD HAPPEN? Finally, the ethanol is used to solidify the DNA. In water, the DNA will go back to a liquid form, meaning DNA is soluble in water. When it is in ethanol, it uncoils and precipitates (becomes a solid) leaving behind the other cell components that are not soluble in ethanol. MATERIALS (Items with * are on your lab tray). Solutions will already be prepared. *test tube rack *2 corks or caps *test tube brush *Microscope *Microscope slide/Cover slip *buffer solution *inoculating loop *source of DNA -57 g granulated sugar *source of DNA *blender - 3 g Epsom Salts *100 ml, 250 ml beakers - distilled water to 500 ml *Mortor & Pestle *Liquid detergent solution *Pipettes -10% detergent solution *graduated cylinders (10 ml and 100 ml) 90 ml water to 10 ml dawn *distilled water *Salt Solution *95% ice cold ethanol (poisonous) - 29.2 g salt, distilled water *test tubes PROCEDURES for a basic DNA extraction (edit to fit your actual lab) 1. Cut off a section of your ―living‖ substance (1 inch square) and place on a dish. 2. Combine 100 ml prep buffer and 10 ml detergent, and DNA source solution in blender. *Rinse graduated cylinders after each use. 3. Blend for 1 minute or until mixture is smooth. 4. Pour the mixture into a clean beaker. 5. Transfer 2ml of mixture, not the foam, to a test tube. 6. Add 2 ml of salt solution, screw on cap/cork, and shake vigorously for 10 minutes. 7. After shaking for 10 minutes, look at mixture and note the layers: a. pellet (solids) will float and be in upper layer (looks like foam) b. lower layer is usually called the liquid or supernatant and has DNA. 8. Carefully pour off the lower liquid layer into a clean test tube with a pipette. Make sure the solids stay in the first tube. 9. Use a 5ml pipette pump to slowly pour 5ml ice cold ethanol carefully down the side of the tube with the liquid, not the pellet, to form two layers of liquids. 10. Let the mixture sit undisturbed for 2 minutes. 11. The DNA will float in the alcohol. The DNA should easily spool (collect). 12. Get a microscope, slide, and cover slip. 13. Use an inoculating loop to spool your DNA. 14. Take a small sample of the DNA and put it on the slide and place a cover-slip over slide. Observe under microscope. Draw, label and describe—follow microscope drawing protocol. 15. Clean up and have your teacher check your lab station.