DNA Extraction from Wheat Germ Materials Raw wheat germ – 1 level teaspoon Liquid detergent - 1 ml Ethyl Alcohol (95%) – 15 ml (COLD) on Ice 50o – 60o Celsius tap water – 20 ml Hot plate with Beaker of Tap Water Thermometer 2 - 50 ml beakers 1 - Graduated cylinder (large) Glass stirring rod for stirring mixture Paper towel or Eye dropper – may be needed to remove foam Paper clip hook or wooden stick or spaghetti for collecting DNA PROCEDURE: 1. Place 1 LEVEL teaspoon of raw wheat germ in a 50 ml beaker 2. Add 20 ml of hot (50-60 °C) tap water and mix constantly for 3 minutes. 3. Add 1 ml or (15 -18 drops) of detergent and mix gently for 5 minutes. DO NOT MAKE FOAM 4. Use a piece of paper towel or an eyedropper to remove any foam from the top of the solution. 5. BEFORE adding the alcohol, POUR the water/detergent solution into a clean beaker, leaving behind the wheat germ. 6. Tilt beaker with the water/detergent solution at an angle. SLOWLY pour 15 ml of COLD alcohol down the side so that it forms a layer on top of the water/wheat germ/detergent solution. Do not mix the two layers together. (DNA precipitates at The water-alcohol interface (the boundary between the water and the alcohol). Therefore, it is crucial to pour the alcohol very slowly so that it forms a layer on top of the water solution. If the alcohol mixes with the water, it will become too dilute and the DNA will not precipitate.) 7. Let the beaker sit for a few minutes. White, stringy, filmy DNA will begin to appear where the water and alcohol meet. You will usually see DNA precipitating from the solution at the water-alcohol interface as soon as you pour in the alcohol. If you let the preparation sit for 15 minutes or so, the DNA will float to the top of the alcohol. 8. You can usually get more DNA to precipitate from the solution by using one of the DNA-collecting tools (paper clip hook) OR spaghetti to gently lift the water solution up into the alcohol. This allows more DNA to come in contact with the alcohol and precipitate. Then use paper clip hook or a wooden stick or spaghetti to collect the DNA. 9. Place DNA on paper towel and air dry. FEEL IT. 0R 10. If time permits, place a small sample of DNA on a slide with coverslip; look at it with microscope. Be sure to share results with all lab partners!!! Teacher Signature: CLEAN UP !!!!! DNA EXTRACTION Group Names: ______________________ ______________________ ______________________ ______________________ Name________________ Table#_____ Date________________ Per._____ 50% of your Grade is the DNA Product Teach sign READ NOTES ON BACK 1. List three things available to you that you might use for a DNA source besides wheat germ, onion, cheek cells or split peas. 2. List three things that would not be a DNA source. 3. What is the role/purpose of DNA in a cell? 4. a. Explain how DNA is contained in a cell. b. Why does DNA extraction need to be performed in order to obtain DNA? 5. a. Describe what you saw happening in the beaker. b. Describe the product that was obtained. 6. What is an Enzyme? 7. Why did you use detergent in the procedure, and how does it work? (see notes on back) 8. What possible benefits might be obtained by the ability to isolate the DNA of any organism? 9. Which “ingredient” (besides wheat germ,cheek cells, onions, or peas) do you think might have the most effect on the amount of DNA you extract? __________________________________________ 10. How can you test your Hypothesis? Describe an experiment to test your hypothesis. HOW DOES IT WORK? DNA is present in all living things from bacteria to plants to animals. In animals, it is found in almost all cell types: cheek, muscles, reproductive cells, hair roots, -- anything with a nucleus. DNA is NOT found in Red blood cells because they lack nuclei. White blood cells do have a nucleus. DNA in a cell is about 100,000 times as long as the cell itself. However, DNA only takes up about 10% of the cell's volume. How can this be? This is because the DNA molecules fold themselves many times to pack themselves in the cell's nucleus. Each chromosome contains a single immense molecule of DNA that, in humans, has a length of up to 12 centimeters when stretched out! (look at 12 cm on ruler) As a matter of fact, all the DNA in one human cell (on all 46 chromosomes) is about two meters long, yet fits into a cell nucleus which is 2-3 micrometers (that's .000002 meters wide!). Yet, the DNA must still be in such a state as to allow for enzymes to replicate the molecule or initiate the production of a protein. The 23 pairs of human chromosomes are estimated to include about 100,000 genes. WHEAT GERM Wheat germ comes from wheat seeds. The “germ” is the embryo, which is the part of the seed that can grow into a new wheat plant. When wheat seeds are milled into white flour, the wheat germ and wheat bran are removed, leaving only starch. Wheat germ contains many nutrients while wheat bran consists of fiber. Whole-wheat flour contains all parts of the wheat seed and is therefore more nutritious than white flour while also providing important fiber for digestion. CHEEK CELLS Cheek cells come from the inner lining of mouth or the check. These cells are routinely shed and replaced by new cells. As the old cells die, they accumulate in the saliva in the mouth and can easily be collected by using mouthwash. One might say “Spit Happens”. ONION CELLS An onion is used because it has a low starch content, which allows DNA to be seen. The walls of onion cells are made of cellulose, which is a polysaccharide made of glucose. Cellulose provides a tough barrier that protects the cell. In order to examine the contents of a plant cell, blending helps to burst open the cell's walls and release contents. Onion cells also have membranes that envelop their contents. Cell membranes are made of a double layerof lipids that allow the movement and transfer of certain ions and substances in and out of the cell. Breaking down a cell's membrane allows you examine the contents of the cell. Detergents contain chemical compounds that can break down cell membranes. Water temperature The heat softens the phospholipids (fats) in the membranes that surround the cell and the nucleus. It also inactivates (denatures) the deoxyribonuclease enzymes (Dnase) which, if present, would cut the DNA into such small fragments that it would not be visible. Denatured enzymes and DNA unravel, loose their shape, and thus become inactive. Enzymes denature at 60°C and DNA denatures at 80°C. Detergent Detergent contains sodium laurel sulfate, which cleans dishes by removing fats and proteins. It acts the same way in the DNA extraction, pulling apart the fats (lipids) and proteins that make up the membranes surrounding the cell and the nucleus. Once these membranes are broken apart, the DNA is released from the cell. Soap molecules and grease molecules are made of two parts: Hydrophilic heads: which LIKE water and Hydrophobic tails which HATE water Both soap and grease molecules organize themselves in bubbles (spheres) with heads outside to face the water and tails inside to hide from the water. When soap comes close to grease, it captures it, forming a greasy soapy ball: A cell’s membrane has two layers of lipid (fat) molecules with proteins between them: When detergent comes close to cell, it captures the lipids & proteins & releases DNA: Alcohol The DNA released from the cell nucleus is dissolved in the water/detergent/wheat germ solution and cannot be seen. DNA precipitates out of solution in alcohol, where it can be seen. Besides allowing us to see the DNA, the alcohol separates the DNA from the other cell components, which are left behind in the water solution. The alcohol also causes gases dissolved in the water to be released, which may be observed as small bubbles. Meat Tenderizer : acts as an enzyme to cut proteins just like a pair of scissors. The DNA in the nucleus of the cell is molded, folded, and protected by proteins. So, the enzyme (papain) cuts the proteins away from the DNA. Papain also helps break down DNAase, an enzyme that breaks down DNA) (Not used in all of the DNA extractions) Results Questions: 1. What does the salt do? (gatorade) (Salt provides the DNA with a favorable environment; it contributes positively charged atoms that neutralize the normal negative charge of DNA.) 2. What does the blender do? (help break down the cell walls) 3. When you mix the blended cell source with the soap, what is happening? (In the experiment, the enzymes in the soap are breaking down the lipid molecules of the cell and nuclear membranes, releasing the contents of the cell, including the DNA. These enzymes in the soap are what break down grease while washing dishes.) 4. What does the alcohol do? Why does the DNA rise to the top after adding alcohol? (DNA will not dissolve in this alcohol, so the DNA comes out of the solution, or precipitates. It is less dense than water or cell scum--which is what settles to the bottom of the glass--so it floats up into the alcohol layer, where you see it as a snotty, string-like substance, with small bubbles formed on it.) 5. If you try a seed food such as peas, there will be more protein residue in the liquid. Why? (Because protein is stored in them for the nutrition of the new plant.) 6. Why can’t you see the double helix? (It is too small to be seen with the naked eye. What you extracted is millions of strands of DNA.) 7. What part of the cell did the DNA come from? (99% is from the nucleus.) BONUS: 1. If you did the experiment with both plant and animal cells, how would their DNA compare?