DNA Extraction Kit. I. What is DNA?

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1 DNA Extraction Kit I. What is DNA? We all know that dogs only give birth to dogs, giraffes to giraffes and so on for every type of living creature. But why is this so? The answer lies in a molecule called deoxyribonucleic acid or DNA, which contains the biological instructions that make each species unique. DNA, along with the instructions it contains, is passed from adult organisms to their offspring during reproduction. It is the hereditary material in humans and almost all other organisms. Most DNA is located in the cell nucleus, but a small amount of DNA can also be found in the mitochondria. The information in DNA is stored as a code made up of four chemical bases: adenine (A), guanine (G), cytosine (C), and thymine (T). Human DNA consists of about 3 billion bases, and more than 99 percent of those bases are the same in all people. The order, or sequence, of these bases determines the information available for building and maintaining an organism, similar to the way in which letters of the alphabet appear in a certain order to form words and sentences. DNA bases pair up with each other, A with T and C with G, to form units called base pairs. Each base is also attached to a sugar molecule and a phosphate molecule. Together, a base, sugar, and phosphate are called a nucleotide. Nucleotides are arranged in two long strands that form a spiral called a double helix. The structure of the double helix is somewhat like a ladder, with the base pairs forming the ladder s rungs and the sugar and phosphate molecules forming the vertical sidepieces of the ladder. (1) References 1. National Library of Medicine (US). Genetics Home Reference [Internet]. Bethesda (MD): The Library; 2014 Oct 9. What is DNA?; [cited 2014 Oct 14]; Available from:

2 II. DNA Extraction kit 1. Introduction: Have you ever wonder how DNA looks like in real life? This kit helps that come true. Especially, you can see your DNA (From cheek cells) by naked eyes. Besides, this kit also introduce to you three basic steps applied in all DNA extraction experiments. Those are break down cell membranes, removing proteins and precipitation of DNA. The following is the content of the kit: _ Alcohol.. 10ml _ Proteinase... 10ml _ Detergent. 10ml _ Salt... g _ Dye 10ml _ Cup 1 _ Toothpick.. 1 _ Filter paper. 2 pcs Step 1 Mix salt solution + sample* Step 2 Filter the solution (If you use fruit as a sample) Step 3 5 drops of Detergent Step 4 3 drops of Proteinase Step 5 20 drops Cold Alcohol ** (+ Dye: If needed) 2. Method: Figure 2: General Procedure for Extracting DNA Time for each step is at least 1 minute. Stir the solution gently after step 3 and 4. After step 5, please wait at least 3 minutes to see if you get DNA or not. (White strings with bubble attached rising through the alcohol layer.)

3 *: You can use human cheek cells, banana, strawberry (The fruit samples must be mashed before mixing with saline solution) **: Put alcohol bottle into the fridge at least 10 minutes before doing the experiment. 3. Some tips: Using distilled water for getting the best results. Making salt solution: 2 teaspoons of salt dissolved in 1/4 liter of water. Alcohol must be kept cold. The colder the alcohol, the higher successful experiment. It is because colder temperatures slow down enzymes that can break down DNA, giving better extraction results. Storage: Always keep the Proteinase bottle into the fridge (If do not use) to maintain the function of it. In extracting DNA from human cheek cells experiment, it is important not to eat, drink, smoke, brush your teeth, chew gum, or use mouthwash for at least 1 hour before taking the samples. Besides, before doing this experiment, you need to chew your cheeks and don t swallow them. Then, violently swish the saltwater in mouth (do not swallow) for 1 minute, making sure to rub tongue along cheeks. You need to do these because more vigorous swishing washes more cells off and yields more DNA. You are also washing bacterial cells from the inside of the mouth, so will isolate their DNA as well. III. Questions and Answers: 1. How does the kit work? _ The detergent removes the greasy cell membranes from around the cells. The cells also have a membrane around the nucleus, which is also removed. The cell contents (including the DNA) are released into the detergent solution. _ Saltwater helps remove cells from your samples and also makes the watery solution denser this increased density will facilitate the separation of the DNA strands into the alcohol. In addition, to get the DNA to precipitate and solidify, we need to do something about each DNA molecule's negatively-charged phosphate backbone. When we dissolve the table salt (NaCl) in the solution, some of the positively-charged sodium ions will interact with the negatively-charged regions of the DNA molecules and effectively shield other nearby DNA molecules from their repulsive force - this will help them all aggregate and clump together. To visualize the idea here, image the resistance you feel when you begin to push the north poles of two magnets together this is sort of like what s going on between the individual DNA molecules. Now image inserting the south pole of a third magnet between the north poles of the first two the resistance is reduced. The south pole of the third magnet is sort like the sodium ion in our solution.

4 _ Proteinase is a type of enzyme that can break down proteins. A tiny bit of this enzyme should reduce the amount of protein that precipitates out with your DNA later on. _ Alcohol is less dense than water so it floats on top of the water. Most of the cellular components are heavy (dense) enough to remain in the watery solution at the bottom of the test tube. DNA, however, is less dense than either the water or the alcohol, so it floats to the surface of the alcohol. DNA is also soluble in water but precipitates in alcohol. _ DNA is exceedingly thin but clumps together. This is similar to the idea that cells are microscopically small (invisible to the naked eye) but clumped together in a multi-cellular organism, are quite visible without the need for a microscope. 2. I don't think I'm seeing DNA. What should I be looking for? Your DNA may be lingering between the two layers of alcohol and water. Look very closely at the alcohol layer for tiny bubbles. Even if your yield of DNA is low, clumps of DNA may be loosely attached to the bubbles. 3. What can I do to increase my yield of DNA? Allow more time for each step to complete. Keep it cold. Using ice-cold water and ice-cold alcohol will increase your yield of DNA. The cold water protects the DNA by slowing down enzymes that can break it apart. The cold alcohol helps the DNA precipitate (solidify and appear) more quickly. Do not use a lot of water in extracting DNA from fruits. 4. Why would a cell contain enzymes that destroy DNA? These enzymes are present in the cell cytoplasm (not the nucleus) to destroy the DNA of viruses that may enter our cells and make us sick. A cell's DNA is usually protected from such enzymes (called DNases) by the nuclear membrane. 5. Is my extracted DNA pure? No, your DNA is not pure. It can mix with something such as proteins, RNA or DNA of some bacteria because this kit includes simple chemical substances and we just want to introduce to you three basic steps (Break down cell membranes, removing proteins and precipitation of DNA) in all extracting DNA experiments. To get the pure DNA, you need to do further steps. 6. Why doesn t my DNA look like the double helix I see in textbooks?

5 The DNA you got is a clump of thousands of DNA molecules. The double strands of one DNA cannot be seen by naked eyes because they are too small to see. 7. How can we confirm the white, stringy stuff is DNA? There is a protocol that would allow you to stain nucleic acids, but the chemical used would need to be handled by a teacher or an adult. So, for now, you'll just have to trust that the molecules precipitating in the alcohol are nucleic acids. You can test its acidity with some natural ph indicators or litmus paper. 8. How long will my DNA last? Will it eventually degrade and disappear? Your DNA may last for years if you store it in alcohol in a tightly-sealed container. If it is shaken, the DNA strands will break into smaller pieces, making the DNA harder to see. If it disappears it's likely because enzymes are still present that are breaking apart the DNA in your sample. 9. Can I use a microscope to see the DNA that I extract? Unfortunately, a microscope will not allow you to see the double helical structure of the DNA molecule. You'll only see a massive mess of many, many DNA molecules clumped together. In fact, the width of the DNA double helix is approximately one billionth of a meter! This is much too small to see, even with the most powerful microscope. Instead, a technique called X-ray crystallography can be used to produce a picture of the DNA molecule. Anyway, you should stick them under microscope to see how they look like and to satisfy your curiosity. Don t be hesitated because this is science! It requires your curiosity to discover new things. However, you must transfer DNA strands very carefully as they are extremely fragile. Whatever you do though, make sure you dispose of the results properly. Don't drink your DNA, or sell it off to a strange-looking chap in a dirty white lab coat who mentioned something about cloning. ^^ 10. How is DNA extraction useful to human beings? The extraction of DNA from a cell is often a first step for scientists who need to obtain and study the function of a particular gene. Thus, they can use it to diagnose a genetic disease from one person or to mass produce a gene or protein important for treating a disease. In addition, DNA extraction is very useful in finding criminal or identifying blood relationship. IV. Some interesting facts about DNA

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