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DNA Extraction Lab Purpose: To provide you with the opportunity to isolate and observe DNA. You will gain an understanding of how a fractionation procedure is carried out, the roles of each substance involved and how easy it is to isolate DNA. Background: The process of isolating DNA from a cell is the first step for many laboratory procedures in biotechnology. One 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. A solution is made of check cells treated with salt, buffer and detergent (SDS). The salt shields the negative phosphate end of DNA which allow 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. The buffering agent maintains the pH of the solution so that the DNA stays stable. Collectively, the salt solution and detergent are referred to as the lysing or homogenization buffer. Protease, an enzyme that digests proteins, is added to remove proteins bound to the DNA and to destroy cellular enzymes that would digest the DNA. Materials and Equipment 3ml water (in 15ml tube) protease solution lysis buffer ice cold ethanol 3 disposable pipets Procedure 1. Obtain 3ml of water and label with your initials. 2. Gently chew the insides of your checks of 30 seconds. DO NOT draw blood!! 3. Take the water from the 15ml tube into your mouth. And swish the water around vigorously for 30 seconds. 4. Expel the liquid back into the 15ml tube (there are also some small cups available to help facilitate the transfer. 5. Add 2ml of lysis buffer to your tube. 6. Place the cap on the tube and gently invert the tube 5 times (do not shake the tube). Observed your tube. Any changes? 7. Add 5 drops of protease to your tube. 8. Place the cap on your tube and gently invert it a few times. 9. Place your tube in a test tube rack or beaker in the water bath and incubate at 50oC for 10 minutes. 10. Holding your tube at a 45o angle, fill you tube with cold alcohol by adding approximately 10ml to your tube. It will take repeated additions to add 10ml using the disposable transfer pipet. 11. Place your cap on your tube and let it site undisturbed for 5 minutes. Observe and note any changes in your preparation. 12. After 5 minutes, slowly invert the tube 5 times to help the DNA, which has begun to precipitate, to aggregate. Questions 1. What is the purpose of the homogenization buffer? Describe the role of the different chemicals. 2. Describe the appearance of the DNA precipitate from step 12.