ANSWERS TO STUDENT QUESTIONS 1. Buffer: A solution that will maintain a constant pH, chelate metal ions and generally maintain an environment that will prevent disruption of the experiment. Precipitate: The solid which is visible when a substance becomes insoluble from a solution. Filter: To separate by passing a mixture through a selective membrane. Emulsify: To physically break up lipid into smaller lipid globules. 2. To maintain an environment in which the DNA will not be degraded and to help separate the DNA from other cell components.
Tris maintains a constant pH. EDTA chelates metal ions. NaCl provides Na+ ions that will block negative charge from phosphates on DNA. 3. Negatively charged phosphates on DNA cause molecules to repel each other. The Na+ ions will form an ionic bond with the negatively charged phosphates on the DNA, neutralizing the negative charges and allowing the DNA molecules to come together. 4. Any non-starchy vegetable should work. Potatoes would not work well. 5. Answers will vary. 6. DNA in cells will absorb UV light. When it does it can be damaged.
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If the damage occurs in a cell cycle gene, then growth may become uncontrolled leading to skin cancer. STUDENT PAGE 1 DNA Isolation from Onion INTRODUCTION The process of isolating DNA from a cell is the first step for many laboratory procedures in biotechnology. The scientist must be able to separate the DNA from the unwanted substances of the cell gently enough so that the DNA is not broken up or shredded. The procedure you will be doing is a modification of the “Marmur” preparation which is used worldwide in biotechnology laboratories.
A “Filtrate” is made of onions treated with salt, distilled water and detergent(SDS). An onion is used because it has a low starch content which allows the DNA to be seen more clearly. The salt shields the negative phosphate end of DNA which allows these ends to come closer so they can precipitate out of a cold alcohol solution. The detergent causes the cell membrane to breakdown by emulsifying the lipids and proteins of the cell and disrupting the polar interactions that hold the cell membrane together. The detergent then forms complexes with these lipids and proteins, causing them to precipitate out of solution.
Collectively, the salt solution and detergent are referred to as a lysing buffer. DNA Extraction Detergent/Salt Solution Add 20 ml of detergent to 20 g non-iodized salt and 180 ml of distilled water. The detergent salt solution is used to break down the fat and proteins that make up the cell membrane. The sale causs the phosphate ends of the DNA to come closer together which will make it easier to precipitate out of solution. | Meat Tenderizer Solution Add 5 g of meat tenderizer to 95 ml of distilled water. Enzymes in the meat tenderizer will break down proteins| Ethanol Solution
Add 5 ml of distilled water to 95 ml of ethanol (alcohol). Rubbing alcohol can be substituted. Place this solution on ice, the colder the better. Alcohol is used to precipitate the DNA. Because DNA is soluble in water, alcohol (ethanol) causes the DNA to precipitate and come out of the solution. DNA will rise into the alcohol layer. | The process of extracting DNA from a cell is the first step for many laboratory procedures in biotechnology. The scientist must be able to separate DNA from the unwanted substances of the cell gently enough so that the DNA is not broken up.
It is both interesting and important to understand the reason for some of the steps in the procedure below. An onion is used because it has a low starch content, which allows the DNA to be seen clearly. The salt shields the negative phosphate ends of DNA, which allows the ends to come closer so the DNA can precipitate out of a cold alcohol solution. The detergent causes the cell membrane to break down by dissolving the lipids and proteins of the cell and disrupting the bonds that hold the cell membrane together.
The detergent then forms complexes with these lipids and proteins, causing them to precipitate out of solution. DNA is a nucleic acid located in the cell’s nucleus. It is found making up the genetic material and is bound to several types of proteins. The nuclear and the cell membranes are a tough protective barrier, made of lipids and proteins, which need to be eliminated in order to release the DNA. It is very important that the directions be followed carefully to ensure good results.