DNA Extraction Materials for all trials • • • • • • • • • • • • • • • • • • • • • • • Sodium Chloride Enzymatic contact lens cleaner Graduated Cylinder Fresh tomato Stirring straw Wheat germ Magnetic mixer Magnetic mixer capsule Lab motor Funnel Your mouth Knife Blender ½ teaspoon (2.5 ml) salt Dishwashing liquid (Dawn) Hot water Small test tube Beaker Beaker lid with filter one bottle 95% cold isopropyl alcohol (cold from the freezer) DNA hook Distilled water Toothpicks Procedure for wheat germ extraction 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Place one teaspoon of raw wheat germ in a beaker. Add 20mL of hot (60 degrees C) distilled water and mix constantly with stirring straw for 3 minutes. Add 1mL of dishwashing soap; mix gently with stirring straw constantly for 5 minutes. Try not to create foam. Use a toothpick to remove any foam from the top of the solution. Tilt the beaker at an angle. Slowly pour 14mL of alcohol down the side of the beaker so that it forms a layer on top of the water/wheat germ/detergent solution. Do not mix the two layers together. Let the beaker sit for 15 minutes. This will let the DNA float to the top. Use the DNA hook to collect the DNA and store in a small container with 70% cold isopropyl alcohol. Analyze the DNA and record observations about the entire process and DNA in the data table. Hypothesis As I extract different types of DNA, then the difficulty of extracting the wheat germ will be hardest because the wheat germ had been dried. Problem How does the different types of DNA affect the simplicity of its extraction, and which DNA will be the hardest to extract? Variables Manipulated Variable: type of DNA (form where the DNA was extracted). Responding Variable: observations on which DNA was extracted easiest Controlled Variable: Type(s) of DNA, volume of alcohol, volume of wheat germ, volume of tomato solution, extracting tools, size of test tube, percent of isopropyl alcohol, size of beaker. Procedure for Tomato Extraction 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Take a 200g fresh tomato and place in the blender. This will be the source of the DNA. Add a large pinch of sodium chloride (less than 1mL or 1/8 of a teaspoon). Add 400mL of cold distilled water. Blend on high for about 30 seconds, or until thoroughly blended. Pour the thin tomato-cell soup mixture through the beaker lid with the filter into the beaker. Add about 1/6 of the amount of tomato mixture of dishwashing soap. Put the magnetic mixer capsule into the beaker, and place the beaker onto the magnetic mixer base. Place the magnetic mixer base lid on the magnetic mixer base. Mix on high for 1 minute. Let mixture sit for 5-10 minutes. Pour mixture into the test tube using the funnel. Fill the test tube about 1/3 full. Add 3 drops of the enzymatic contact lens cleaner to the test tube. Stir gently. Fill the graduated cylinder with the isopropyl alcohol and gently pour the alcohol down the side of the test tube while you hold the test tube at an angle. Pour until you have the same amount of alcohol in the test tube as the tomato mixture. DNA will rise into the alcohol layer from the tomato layer. Use the DNA hook to remove the DNA and store in a container with 70% isopropyl alcohol. Analyze the DNA and record observations about the entire process and DNA in the data table. Procedure for Human DNA Extraction 1. Dissolve ½ teaspoon of sodium chloride in 50mL of room temperature distilled water in a beaker. 2. Add 2mL of dishwashing soap. 3. Take 25mL of distilled water into mouth. Do not swallow. 4. Swish water around mouth vigorously for 30 seconds. 5. Spit the water into a clean beaker. 6. Add 5mL of this spit fluid to a test tube with a cap. 7. Add 2.5mL of the salt/dishwashing liquid solution to the test tube with the spit fluid. 8. Put the cap on the test tube and tip it up and down gently 4 times to mix (don’t shake). 9. Gently run a teaspoonful of cold isopropyl alcohol into the tube. 10. Wait 10 minutes. 11. Analyze the DNA and record observations about the entire process and DNA in the data table. Background Information DNA, or deoxyribonucleic acid, is basically the instructions for the cells in the body of any living being. In the nucleus, which is basically the brain or control room of every cell, there are strands of these instructions on how to make the cell perform a certain action such as making a specific protein. But DNA can mutate. Even one simple mutation in the DNA can have extreme effects such as sickle cell disease. DNA is even found in some viruses. A strand of DNA contains chemicals called nucleotides. A DNA molecule is made up of 2 polynucleotide chains arranged on the double helix (the backbone). These nucleotides are composed of three parts: a phosphate, a sugar (deoxyribose), and a type of compound base. The amount of each nucleotide and the how they are arranged is different for all living things. The ways to organize the nucleotides are endless, and that's why everybody in the world looks different. 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, kind of like the way in which letters of the alphabet appear in a certain order to form words and sentences. The bases are connected together to form a base pair, and the bases can only be paired off in a specific way. An adenine base on one side of the chain only bonds with a thymine base on the opposite side, and a guanine base can only bond with a cytosine base. Or you can look at it this way: A=T and G=C . History of DNA: in 1865 Gregor Mendel discovered the fundamental laws of inheritance. He experimented with pea plants between the years 1856 and 1863. Mendel used over ten thousand pea plants and kept track of all of the This is there first real experiments with DNA or genes. From then on, many people made amazing discoveries with genes and DNA. Mutations were discovered. There were many advancements in DNA knowledge. Mutations in DNA sequences normally occur through one of two processes: DNA damage from environmental agents such as ultraviolet light (sunshine), nuclear radiation or certain chemicals , or mistakes that occur when a cell copies its DNA in preparation for cell division. Ultraviolet light, nuclear radiation, and certain chemicals can damage DNA by altering nucleotide bases so that they look like other nucleotide bases. When the DNA strands are separated and copied, the altered base will pair with an incorrect base and cause a mutation. Environmental agents such as nuclear radiation can damage DNA by breaking the bonds between oxygens (O) and phosphate groups (P). Breaking the phosphate backbone of DNA within a gene creates a mutated form of the gene. It is possible that the mutated gene will produce a protein that functions differently. Cells with broken DNA will attempt to fix the broken ends by joining these free ends to other pieces of DNA within the cell. This creates a type of mutation called "translocation." If a translocation breakpoint occurs within or near a gene, that gene's function may be affected. Diagram This is part of the structure of a DNA double helix. Data Table of Observations Wheat Germ • Filmy, milky in color • Appears as bubbles or chunks • Easy to extract • Also small strings • Easy process and appears quickly Tomato Human DNA •Not very much • Small, hard to see DNA • Takes several •Takes an average minutes to form amount of time to • Fragile thin lines, form white in color •Appears as a stringy pink layer • Hard to extract between alcohol • Longer process and and tomato mixture a longer time to •Strands of tissue appear •fragile Abstract 1. The purpose of this project was to determine whether different types of DNA would affect how easily the DNA was extracted, and which of the three types would be the hardest to extract. 2. I believed that different types of DNA would differ in how easily they were extracted and that the wheat germ’s DNA would be the most difficult. 3. A. There were no major errors in this experiment. B. After the completion of my experiment(s), I learned that it was in fact more difficult to extract different types of DNA. The wheat germ was actually easiest, and that my own human DNA was the most difficult to extract. Conclusion I thought that as I extract different types of DNA, then the difficulty of extracting the DNA of a wheat germ will be the highest because the wheat germ has been dried. My results proved this incorrect. I can prove this because the lowest amount of effort it took to obtain the DNA was with the wheat germ, when there was no difficulty at all. The highest amount of difficulty it took to obtain the DNA was with the human DNA, when the DNA took several times to show up. The difference between the two was that the wheat germ’s process was simple to follow and the DNA was easy to see, compared to the human’s more complex procedures and hard to see DNA. This rejects my original hypothesis. Therefore if I were to repeat this experiment, the human DNA would be the hardest to obtain, versus the easy wheat germ extraction.