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SBI 4U Mrs. Holterman Name: ________________________ Date: ________________________ DNA Extraction Lab Pre-Lab Questions Read through the rest of the lab to answer the following questions before the lab. 1. Why does the plant tissue (strawberry) have to be heated, but the animal tissue (liver) does not? 2. What are you accomplishing at the cellular level when you: a. Blend the sample? b. Heat the sample? c. Add meat tenderizer (cleaving enzymes)? d. Add detergent? e. Add alcohol? f. Add salt? 3. Why can’t you shake the test tube in step 1? 4. Answer the question in the purpose and give some explanation. 5. Create an observation table. Introduction In this investigation, you will isolate DNA from strawberries and liver. DNA is 100 000 times longer than the cell itself, but only takes up about 10% of the space in the cell. It achieves this by tight folding and packaging with proteins. DNA extraction is the first step in many biotechnological procedures. Cell walls and cell membranes must be disrupted to isolate DNA, using a kitchen blender before the experiment begins. Dish detergent will dissolve the phospholipids in a process called cell lysis. Heat, detergents, salts and cleaving enzymes will be used to further minimize contamination. Purpose Which type of cell has more DNA: plants (strawberries) or animals (liver)? Method Part 1: 1. Add 7mL of the detergent to a small test tube. Then, add 7 mL of the liquefied strawberries to the detergent. Use a rubber stopper to seal the test tube. DO NOT SHAKE!! Mix by slowly turning the test tube up side down. 2. Place the test tube in a 65C water bath for 15 minutes to denature the proteins. (While waiting, begin part 2). 3. Add 7 mL of meat tenderizer solution and swirl to mix. Meat tenderizer is full of protease enzymes. 4. Use a funnel to secure a filter paper in the large test tube. Wet the filter paper with water. Slowly pour the strawberry mixture into the SBI 4U Mrs. Holterman 5. 6. 7. 8. Name: ________________________ Date: ________________________ filter. Let the clear filtrate drip into the test tube (this might take 10 minutes). Hold the test tube at a slight angle and pour in a matching volume of ice cold ethanol on top of the filtrate using a pipette and letting it run down the side of the tube. ** Don’t let it splash! ** Since DNA is insoluble in cold alcohol, it should appear at the interface of the detergent and the alcohol, as a cloud of fine threads. Take observations of this DNA. If your DNA is not precipitating out of the ethanol, add a pinch of salt. If you want to remove the DNA from the solution, use a wooden splint to spool the DNA onto it by gently twisting the stick. Be careful not to mix the layers. Part 2: 1. Obtain a piece of liver and cut it into small pieces. 2. Place the pieces into the mortar, along with 10 mL of 0.9% NaCl solution. Add a pinch of sand into the mixture to act as an abrasive, and grind the tissue thoroughly for approximately 5 minutes. 3. Place a filter paper into a funnel sitting on a test tube. Pour the remains of the liver and its liquid into the filter paper. Let the filtrate drip into the test tube. This might take 10 minutes. 4. Add 3 mL of detergent to the test tube. Mix well (but DO NOT shake!) for 30s. 5. Hold the test tube at a slight angle and pour in a matching volume of ice cold ethanol on top of the filtrate using a pipette and letting it run down the side of the tube. ** Don’t let it splash! ** 6. The DNA should appear at the interface of the detergent and the alcohol. 7. If your DNA is not precipitating out of the alcohol, add a pinch of salt. Analysis A) Look at your description of the DNA that you extracted. If DNA is a rigid structure, why do the strands appear flexible? What features of DNA’s structure account for its rigidity? B) What chemical properties of DNA does this lab make use of? C) Compare the amount of DNA extracted from the plant to the amount from the liver. Is this what you expected? Why or why not? D) Identify the areas of the procedure that may have lead to an incorrect answer to the purpose. How could you change the procedure to get a more accurate answer?