Extract DNA Experimenter Kit

Did you know

DNA is the longest molecule known and it exists in every living cell. The code it carries determines the form and behavior of all life on earth. When released from a cell, DNA typically breaks up into filaments. In solution, these strands have a slight negative electric charge, which makes for some fascinating chemistry. For example, the more negative sections of one DNA strand will tend to attract the more positive regions of another. This causes these DNA floating molecules to clump together into a big gooey mass that you can easily see. However, if salt is added, the salt ions are attracted to the negative charges on DNA, effectively neutralizing them. This stops the separate fragments from sticking together and keeps them floating about in solution. So by controlling the salt concentration, biologists can make

Age 12+

DNA stands for Deoxyribonucleic Acid

DNA fragments either disperse or glom together. And therein lies the secret of separating DNA from cells. The materials I've provided you in my Super DNA Experimenter's Kit will make it easy for you to extract and purify in your kitchen DNA from living things exactly as professional scientists do in their laboratories. Pretty cool, eh? Sundries Graduated Test Tube to harvest the DNA and measure your yield. Nylon Fast-flow Filter to remove the gunk after you've extracted the organic molecules from the cells. Glass Extraction Rod to remove the DNA from the test tube. Plastic 4 oz. squeeze bottle to conveniently hold and dispense rubbing alcohol. Three 250 ml Tri-pour Beakers to mix and process the materials. Not included Rubbing or Isopropyl Alcohol: Isopropyl alcohol is a hazardous material and is far too expensive to ship. Use the highest concentration that your drugstore sells. NOTE: You MUST chill your alcohol in the freezer before you begin!

Your Kit Contains

For the buffer solution Powdered Buffer: Contains 1) DNA Suspension Agent 21.5% NaCl (pure table salt) to lubricate the DNA molecules to keep them from sticking together. 2) pH Stabilizer: 77.3% NaHCO3 (purified baking soda) to neutralize any acids in the organic matter. 3) Protein Destroyer: 1.2% Papain enzyme, eats proteins so they don't contaminate your DNA. Cell Blaster: sodium dodecyl sulfatea detergent that rips up cell walls and nuclei so the molecules inside can seep out.

To Stain the DNA Blue Monster DNA stain to make the DNA clearly visible.

Page 2 of 7

Step One: Mix the Buffer

Definition: In chemistry, a "buffer" is a solution that tends not to change its acidity, or pH, during an experiment. It protects, or "buffers," a reaction against changes in pH.

Why not use tap water?

Tap water isn't pure. It almost always contains all sorts of chemicals that could ruin your results. In particular, tap water is often loaded with calcium and magnesium ions that can interact with the detergent, and chlorine that destroys DNA on contact. Bottled water, while not perfect, works great for this experiment. However, distilled water works best, so use that if you can. You can buy it at your local grocery. DNA Fact: DNA exists
in several different parts of a cell. The "genomic DNA" lives in the cell nucleus and contains the complete genetic recipe of the organism. Only the genomic DNA molecules are large enough to be extracted by the simple procedure described here.

You'll first need to whip up your buffera solution into which DNA can float about. Here's how Add water: Pour 120 milliliters (about 4 ounces) of distilled or bottled water into one of the beakers provided. Add the Powdered Buffer: Carefully measure 1 and 1/4 tsp Mix until completely dissolved before going on to the next step! The powder will not fully

dissolve after you add the detergent. Dont believe me? Try it! Add detergent: 5 milliliters (1 tsp) of Dr. Shawn's super Cell Blaster. Mix gently to avoid suds. Chill the Buffer: DNA degrades quickly. So to slow the destruction, briefly chill the buffer in an ice water bath before proceeding. Don't let the buffer turn milky. If it does, warm it in your microwave oven just enough to make it go clear and then chill it again. Optional (increases yield): Heat some water in a Pyrex measuring cup (best in the microwave) until it measures between 130-140 F (5466 C). Be careful! Place about 2 tablespoons of your mush inside a plastic sandwich bag. Remove as much air from the bag as possible and seal it. Then steep the bag for 10 minutes in the hot water, like you would tea. Don't let the bag heat any longer than 15 minutes, however, or the DNA may start to break down. The idea is just to soften up the cell membranes. Also, it should break up the DNAse enzymes that are present in the cells that chop DNA up into small fragments. Cell Lysis: Place 15 milliliters (1 Tbs) of the mush into a clean beaker. Mix in 30 milliliters (2 Tbs) of your chilled buffer. Swirl gently for at least two minutes. In this step, the detergent breaks down both the cell walls and most important, the membranes around the cell nuclei, where the genomic DNA is located. Without it, the genomic DNA would never get out of the nuclei and into the buffer.
Warning: If you agitate this molecular soup too vigorously, you'll break up the DNA into tiny fragments. If that happens, the DNA pieces will still fall out of solution into a visible mass, but you wont be able to get them to spool like spaghetti onto the glass extraction rod.

Step Two: Get the DNA

For a source of DNA, try the pantry. Onions give great results. Garlic, bananas and tomatoes work well too. But this is your experiment. Choose your own personal favorite fruit, vegetable, fungus, or legume. You can even try meat or bone marrow from soup bones. (Tip Try cows tongue. It looks totally disgusting, but youll get great results!) Or try the leaves of your favorite tree, or something growing outside. It doesn't matter what you try, so long as it is something that interests you! Once you've got your DNA source, you'll need to process its cells to get out the DNA. Here's how: Prepare the bulk matter: Dice the material into chunks using a kitchen knife. Mash it up: Put the material into a blender or food processor. Pour in just enough distilled or bottled water to cover the chunks. Expose the cells: Break up the cells by pulsing the blades until the material has been converted into a slushy mush. These treatments will break apart some of the cells right away and expose many more cell membranes to attack by the detergent.

DNA Fact: Genomic

DNA is the largest molecule known. A single strand can contain millions of atoms.

Page 3 of 7

Step Three: Separate the Fluid From the Solids

Filter: Next you must separate the cellular debris from the moleculeladen soup. To do so, use the fastflow nylon filter included with this Experimenter's Kit. The fine nylon mesh makes a wonderful filter. Stretch the open end over a clean beaker. Let the contents stand and drain on their own for about 30 seconds or so until the fluid has stopped dripping. Then discard. When youve completed your experiment, dont forget to gently wash the filter in warm soapy water. If properly cared for, your filter should last a very long time.

Warning: Do not force the fluid through the filter by squeezing it with your hand. If you do, you will drive some of the cell matter into the solution and contaminate your DNA. The fast-flow nylon filter makes it easy to separate the cellular matter from the buffer.

When You've Liberated the Liquid From the Cellular Matter

The clear filtered liquid contains the buffer chemicals plus lots of organic moleculesDNA, proteins (which are being broken down by the Protein Destroyer enzyme), RNA, carbohydrates and othersthat leached out of the cells. Remember, the DNA stays dissolved only because the salt ions in the buffer prevent the negatively charged DNA molecules from sticking together. Now, you're ready to reduce the salt concentration to let those molecules clump up and "precipitate" (pre-ci-pi-tate) out of the solution.

Step Four: Extract the DNA

Transfer 5 milliliters (one tsp) of the filtered buffer to the plastic graduated test tube. You should have chilled your rubbing alcohol in your freezer so that it is now ice cold. If not, do so now and toss out your sample. The DNA won't last until its chilled. If so, get the squeeze bottle out of the freezer. Carefully deposit about 5 milliliters of the chilled alcohol on top of the DNA solution by tilting the test tube as shown and gently squeezing the plastic bottle. This will allow the alcohol to stream slowly down along the inside of the test tube so it flows onto the buffer gently. Alcohol is less dense than the buffer and so it will float on top.

Where the two liquids meet, a gelatinous sludge should appear.

That sludge is DNA!

At this point, you should see three distinct layers; the extraction agent (top), the DNA sludge (middle), and the buffer (bottom). NOTE: Both RNA and protein molecules can also be extracted in the same way as DNA. That's why this Experimenter's Kit includes our Protein Destroyer. This enzyme is what scientists call a protease (prote-ase)it destroys protein on contact. However, you'll find no Rnase (enzymes that degrade RNA) here. Why not? Because, believe it or not, RNA-busting chemicals are so abundant in nature and so hearty that they naturally occur nearly everywhere, even in distilled water! In fact, it's hard to prepare a solution

solution that doesn't have them. So your DNA should be nearly pure.

When the Extraction Agent is made to flow gently on to the top of the buffer, a layer of DNA sludge appears between them.

Page 4 of 7
200 Experiments: This research kit contains enough chemicals to make 20 batches of buffer and each batch will let you do at least 10 DNA preps. That means you can do at least 200 trials!

Welcome to the World of Molecular Biology!

This Experimenter's Kit is only your entry visa into the wonderful world of molecular biology. Where you go now depends entirely on you! There are at least two types of explorations that are easy to do at home: those in which one measures the quantity of DNA extracted from different organisms under different circumstances; and those that explore the qualities of DNA itself. Great explorations await you no matter which you choose.

Measuring Quantity
Determining how much DNA you extracted couldn't be simpler. Just read off the thickness of the DNA sludge by looking at the graduations on the test tube that came with your DNA Research Kit. Amount of DNA Per Unit Volume of Living Matter While the volume of DNA produced is a great thing to know, often what you're really after is how much DNA youve extracted from a given amount of material. After all, if you got the same amount of DNA from 5 milliliters of onions as you did from 10 milliliters of oranges, then you could conclude that onions were twice as good a DNA source as oranges. Does that make sense? To make that kind of comparison, you want to divide the volume of DNA you extracted by the amount of material it came from. The simplest way to do that is to accurately measure how much cell mush you put into your buffer, and then process all of the buffer to extract all of the DNA that leached into it. Example: Suppose you put exactly 5 ml of plant mush into 10 ml of buffer and extracted 1 ml of DNA. How much DNA did you get from each milliliter of plant mush? Easy! Just divide what you got by what you started with. 1 ml DNA / 5 ml plant matter = 0.2 ml / ml. (Notice that I've replaced "milliliters" with the scientist's standard shorthand "ml".) NOTE THE UNITS HERE! Since the "ml" units appear in both the numerator and the denominator you may be tempted to cancel them and report this quantity as unit-less. Don't. Reporting your results as "ml/ml" tells the reader that this number expresses the volume of a product that you extracted from a volume of source. Remember, you want your science fair judges to understand at a glance what each of your datum mean. So make sure the info they need clearly labels each calculation and data point. Now, where is the amount of buffer in this calculation? Actually, it's nowhere. After all, who cares how much buffer you put the cell mush in so long as you've gotten all of the DNA out? The buffer merely delivers DNA from the cells to the scientist (that's you!). It acts just like a truck carrying cargo. What matters is the amount of cargo produced and delivered, not the size of the container that carried it. Just make sure to process every bit of the buffer so you get out every bit of the DNA that leached from the cells.

Tip: If you have any questions with the procedure, check out the on-line demo of this project. Go to www.scifair.org and click on the Championship Projects icon. Then scroll down to the Extract DNA In Your Kitchen project and click on the movie icon.

Detail: Why don't we care about the amount of buffer? Because the buffer merely delivers the DNA from the cells to the scientist. It is just like a truck carrying cargo. What matters is the amount of cargo produced and delivered, not the size of the container that carried it.

TIP: Volume is easy to find and is perfectly fine for most science fairs. But mass makes a better measure. If you have access to a sensitive balance, like a lab balance, by all means use it to measure the masses of the processed cellular matter and the extracted DNA.

Dr. Shawn's Experimenter's Kits: Extract DNA in Your Kitchen

Page 5 of 7

Experiments With DNA

While far more challenging than experiments with quantity, you can also experiment with DNA itself. You'll first need to remove the DNA sludge from the original buffer solution that is full of organic contaminants and re-dissolve it in a batch of fresh buffer. Moreover, if you can't complete your experiment in one sitting you'll want to store your DNA for later. Also, it's often useful to stain your DNA to make it easier to see. Here's how to do all of these important steps. How to Remove the DNA First, clean and dry the glass extraction rod thoroughly making certain it is completely free of oils or dirt or chemical contaminants of any kind. You should soap, rub and carefully rinse it in distilled or bottled water and dry it completely with a fresh paper towel. Next, rest the test tube inside a glass so you'll have two hands free. Then gently insert the clean rod through the DNA gunk with its tip just below the boundary of the buffer solution. Hold the rod still with one hand and with the other one very slowly twirl the rod round and round in the same direction. Longer pieces of DNA will spool onto the rod just like spaghetti on a fork, leaving smaller fragments

behind. After a minute or so, pull the rod slowly up through the alcohol. The alcohol will make the DNA adhere to the glass. When you get it into the air you will see a transparent viscous "snot-like" sludge clinging to the rod. There is a bit of artistry required here so don't get frustrated if it doesn't work at first. In fact, usually only a small fraction of your DNA will adhere to the rod. The picture below shows a typical result for a novice molecular biologist, so don't get discouraged. Your results will improve with practice and experience. Re-dissolving DNA You can re-dissolve your DNA in fresh buffer to continue your experiments. Since you aren't extracting DNA from cells at this stage, the fresh buffer need only contain the Suspension Agent and the pH Stabilizer. And remember, less is more here; the less buffer you use the more of the resuspended DNA you will be able to extract later. Store Your DNA For Later Its easy to store your DNA. Just place the viscous sludge that you twirled up on your stick in a container filled with ice-cold isopropyl (rubbing) alcohol, then place the container in the freezer.

Use rubbing alcohol or isopropyl from your local drug store. When stored in chilled alcohol like this, your DNA will keep almost forever. Dyeing DNA Even after the most thorough extraction, some residual DNA will linger in the vessel, forming an invisible cobweb within the liquid. But with a little more effort, you can see that material, too. Dr. Shawn's special Blue Monster DNA Stain, which is included in your Experimenter's Kit, binds directly to charged DNA fragments. A tiny amount added to the remaining solution will thus stain tendrils of uncollected DNA. Add only a tiny droplet: you want all the dye molecules to bind to the DNA, with none left over to stain the water. If you have trouble measuring the quantity of DNA youve extracted, go ahead and drop a droplet of the Blue Monster Stain into the graduated cylinder after you've precipitated the DNA. The droplet will fall straight through the alcohol and cling beautifully to the DNA.

The blue band marks the layer of DNA sludge when stained. DNA extracted from a cow's brain is visible on the end of the Extraction Rod.

Page 6 of 7

Ideas for Science Projects

RememberLearning follows interest. That means that it is hard to learn very much or do very well with projects that don't interest you. So make sure to pick a project that really gets your juices flowing! Doing a science fair project? Then you'll need a question to answer or hypothesis to pose. Here are some ideas for proven championship science fair projects. If none of these appeal to you, that's fine. Learn more and ask your own questions. Doing what interests you is the always the best way to approach any science project. Note: Since most experimenters find it hard to get the DNA out of the test tube, Type One projects are much easier than Type Two. So if you want high certainty of success, stick with Type One.

Type One: Comparing Amounts of DNA Produced Under Different Conditions

Procedure: Extract DNA from different varieties of fruits, vegetables, fungi and other material in your kitchen, garden, butcher shop or the great outdoors. Pick at least three different sources. In addition to fruits and vegetables, try flour, egg whites or egg yokes, hamburger, liver, outdoor plants, and so on. Extract DNA from equal amounts of each one and carefully compare the quantities you find. Questions to consider 1) 2) 3) What kinds of plants yield the most DNA per unit volume: Fruits, vegetables, or legumes? What part of a plant produces the most DNA per unit volume: Flowers, stems, fruits, nuts or leaves? How does temperature affect DNA in vivo (in life)? Use a cooking thermometer to set the temp of water on a stove. Soak whatever youre experimenting with in the bath for several minutes. Remove and chill with ice water. Then find out how much DNA you can extract. Do this at several temps between room temperature and boiling and graph your results. 4) Do organic vegetables yield the same amount of DNA as non-organic? 5) Do fruits yield more extractable DNA when they are underripe, ripe or overripe? 6) Does the fraction of DNA change for different types of fungi, or do all fungi yield about the same amount per unit volume? 7) Try changing the procedure. How much DNA do you get when you use tap water, or use different amounts of detergent, or a different detergent, etc.? Not interested in any of these questions? Then see if they spark any ideas of your own that do interest you!

Type Two: Experiments With DNA Itself d) Finally, reconstitute the DNA in both
Procedure: Here's what you need to do. a) First, extract a large amount of DNA from onions, bananas or another copious source, and store it in isopropyl alcohol in your freezer. b) When you're ready to do your experiment, redissolve a quantity of DNA, say 5 ml (1 tsp), into each of two beakers containing identical amounts of fresh buffer. c) You must treat both samples exactly the same except for just one thing. You expose one sample, your "test" sample, to something that you think might destroy DNA. The other, your "control" sample, you leave alone. Run all samples in pairs side by side. Every test sample must have its own control.

samples by adding isopropyl alcohol. Compare the amount recovered from the test to the control sample to discover how much DNA, if any, was destroyed during your trial. Questions to consider 1) How rapidly does DNA degrade when exposed to sunlight in vitro (in a test tube)? Expose different test samples for different amounts of time to outdoor sunlight. Thin plastic containers let ultraviolet through. 2) How rapidly does DNA degrade at different temperatures in vitro? At a given temperature, plot fraction lost vs. time. 3) How do chemicals affect DNA in vitro? Plot the fraction of DNA recovered when exposed to small concentrations of chlorine (bleach) and other household chemicals.

Resources

Page 7 of 7

Need More Info about DNA? There's tons of great information about DNA online. To find what's current, search "DNA education" on the Internet.

Contact us: Bright Science, LLC 1356 Saxon Lane Naperville, IL 60564 Phone: 630-300-3966 Email: drmichelle@scifair.org

Were on the Web!

For help with any science project:

www.scifair.org

This material is copyrighted by Dr. Shawn (Shawn Carlson, Ph.D.). All rights are reserved.