Activity 1.2.

2: DNA Extraction
Introduction
DNA is the fundamental building block for life. It resides in the nucleus of cells and consists of thousands of genes that contain the instructions for building the different parts of the cell. DNA is what makes us who we are. It determines what physical characteristics we have, such as hair and eye color, as well as some of the diseases we may develop. DNA is a very long molecule, so in order to fit inside the cell, the DNA is highly coiled and twisted into chromosomes. A cell is a complex living unit that contains many biological molecules and organelles, each performing a specific function within the cell. In order to analyze the DNA found at a crime scene, the DNA needs to first be extracted from the cells. This means that it must be separated from all of the other chemicals and materials inside the cell.

Equipment
Computer with Internet access Laboratory journal Inspiration Resource Guide 95% Alcohol (kept ice cold) DNA extraction buffer (well use soap instead) (2) 8cm Squares of cheesecloth (well use a strainer instead) Distilled water Cups of crushed ice Strawberries or bananas Ziploc freezer bags (small, quart size) Small paper or plastic cups (e.g., bathroom cups) 15 mL Screw-cap conical bottom plastic centrifuge tubes Plastic transfer pipettes Glass stirring rod or wooden popsicle stick Knife

Procedure
In this lab you will isolate DNA from plant and animal cells. In order to extract DNA from cells, scientists take advantage of the unique chemical properties of the DNA. Plant cell walls are made of cellulose, which is a tough, insoluble material that makes plants sturdy. It can be broken through physical actions. In this lab you will mash the strawberries in order to break open the cell walls. Cell membranes and nuclear membranes are made up of fats. Detergents wash away these fats, just like they would grease in a pan. In this lab you will treat the mashed strawberry cells with detergent to
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dissolve the cell membranes and nuclear membranes to release the DNA. Once the cells are broken open, the DNA and cell debris are all mixed together. Because the DNA is in solution, you can separate the components by filtering the mixture through cheesecloth (well use a strainer instead). Finally, you can separate the DNA from solution with alcohol, since DNA does not dissolve in alcohol. It is very important that you follow the procedure carefully. Otherwise, your DNA sample will be contaminated with proteins and other cell components. Remember to follow all the safety instructions provided by your teacher. DNA Isolation from Fruit: 1. Put on the appropriate personal protective equipment, including safety gloves and goggles. Tie back any loose hair. 2. Gather the following materials from your teacher. Many items may be located at your station. o 95% Alcohol (kept ice cold) (this will stay in the fridge underneath the front deskget it when you need itit may be 70%-91%, depending on which bottle you grabtheyll all work) o DNA extraction buffer (well use shampoo or dishsoap) o 8cm Squares of cheesecloth (2) (well use a strainer instead) o Distilled water (tap water will work, since were not analyzing this DNA after extracting it) o Cups of crushed ice o Strawberry o Sealable freezer bags (small, quart size) o Graduated cylinder o Paper or plastic cup (well use beakers instead) o 15 mL Screw-cap conical bottom plastic centrifuge tube o Plastic transfer pipette o Glass stirring rod or wooden popsicle stick o Knife 3. Cut the stem off of the strawberry and place into the sealable freezer bag (thats the strawberry youll put inthe stem can be thrown away). Make sure all of the air is out of the bag and mash the strawberry very well. Do not smash the strawberry or the bag will break. 4. Pour 15 mL of DNA extraction buffer into the bag (place a single drop of dish soap inside instead & add up to 10 mL of water along with it if it seems to need more moisture). Seal the bag and mush together for one minute. 5. Place the square of cheesecloth over a small paper or plastic cup. Make sure to create a pocket deep enough to hold the mixture and cover the rim of the cup. Secure with a rubber band if needed. Instead, just dump the strawberry mush into your strainer and put a beaker beneath it
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6. Pour the strawberry mixture slowly into the filter. Make sure NOT to overfill. Pour in only as much as needed to fill the filter at one time. Instead, dump all your strawberry mush in and use your fingers or some other tool to push the liquid through. 7. Once the mixture has filtered through, carefully pour 2 mL of the filtered contents into a clean 15 mL conical tube. You dont have to wait until its ALL filteredjust until you have 2 ml (thats NOT VERY MUCH AT ALL). 8. Hold the 15 mL conical tube at an angle. Using a transfer pipet, carefully and slowly add 5 mL of cold 95% ethanol down the side of the tube. The mixture will sink to the bottom of the tube and alcohol will sit on top. Do NOT mix. Allow the tube to sit undisturbed for two or three minutes. (Id suggest setting it in a beaker) 9. Watch closely as the DNA will appear as a clear-to-whitish, stringy clump where the ethanol layer meets the strawberry extract layer. Tiny bubbles in the ethanol layer will appear where the DNA precipitates. (website people, take a picture!!) 10. Dip the glass stirring rod or wooden popsicle stick into the tube and twirl it around gently to collect some DNA. Stir the DNA gently at the interface between the ethanol and strawberry extract layer to extract more fibers. 11. Carefully remove the stirring rod or popsicle stick from the tube and observe the DNA. Record all observations in your laboratory journal. If you want to take the DNA home, I will give you a centrifuge tube to place it in. Otherwise, you can throw it away after showing it to me. 12. CLEAN UP YOUR AREA BY THROWING AWAY ALL TRASH (used plastic tubes, plastic baggiesall plastic stuff is disposable) and washing/rinsing beakers, etc at the main sink (Ill provide a tub of soapy water). DNA Isolation from Saliva Scientists can use a procedure similar to the one found below to extract DNA from the cells of victims or potential suspects, either in saliva or other bodily fluids. They must be very precise as they work to prevent contamination of the final sample. 1. Gather the following materials from your teacher. Many items may be located at your station. o 95% Alcohol (kept ice cold) o Paper or plastic cup containing salt solution (youll make your own salt solution by dumping some salt into a cup and adding water) o 15 mL screw-cap conical bottom plastic centrifuge tube containing detergent solution o Plastic transfer pipette o Glass stirring rod or wooden popsicle stick 2. Rinse your mouth for one minute with the salt solution. (Make it by adding about a spoonful of salt to a Styrofoam cup and filling it halfway with water the amount of salt isnt TERRIBLY important). Make sure to use your teeth to scrape the inside of your cheeks. This will release the cells into the solution. Spit the salt
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solution containing your cheek cells back into your cup. (when you spit it, spit it into a clean BEAKER). 3. Pour the salt solution with saliva/cheek cells into the tube containing the detergent solution. Gently rock the mixture back and forth for 2 minutes. Instead, place a single drop of dishsoap into your spitty solution and swirl the beaker or stir it with a stir rod for two minutes. THEN place the spitty solution into the conical tube. 4. Hold the 15 mL conical tube at an angle. Using a transfer pipet, carefully and slowly add 3 mL of cold 95% ethanol down the side of the tube. The mixture will sink to the bottom of the tube and alcohol will sit on top. Do NOT mix. Allow the tube to sit undisturbed for two or three minutes. (Id suggest placing it in a beaker). 5. Watch closely as the DNA will appear as a clear-to-whitish, stringy clump where the ethanol layer meets the cheek cell extract layer. Tiny bubbles in the ethanol layer will appear where the DNA precipitates. Website people, take a picture! 6. Dip the glass stirring rod or wooden popsicle stick into the tube and twirl it around gently to collect some DNA. Stir the DNA gently at the interface between the ethanol and strawberry extract layer to extract more fibers. 7. Carefully remove the stirring rod or popsicle stick from the tube and observe the DNA. Record all observations in your laboratory journal. Website people, take a picture! 8. If you want to take the DNA home, I will give you a centrifuge tube to place it in. Otherwise, you can throw it away after showing it to me. 9. CLEAN UP YOUR AREA BY THROWING AWAY ALL TRASH (used plastic tubes, plastic baggiesall plastic stuff is disposable) and washing/rinsing beakers, etc at the main sink (Ill provide a tub of soapy water). 10. Work on the portfolio assignment (see below)

Portfolio Assignment
Complete panel 4 of your portfolio (shown left) by making a matchbook with 3 parts (for the 3 steps of DNA extraction). Label as shown above. Under each flap, write the purpose of that step (review background info in this document and notes from the virtual lab). Be sure to explain what is happening the the cells/DNA in each step.

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