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BIO109 –Laboratory Activity DNA Extraction Purpose: The purpose of this exercise is to extract DNA from a living source – the strawberry and visualize the properties of the purified form of this vital molecule. Introduction: Deoxyribonucleic acid (DNA) can be considered the hereditary “code of life” because it possesses the information that determines an organism’s traits and is transmitted from one generation to the next. DNA can be compared to a list of instructions that tells cells what, when and how much to make a certain product. The DNA in an individual’s cells contains unique genetic instructions about how to make and operate that individual. DNA can be removed from organisms through a common and useful scientific procedure called DNA extraction. In order to understand this process, it is useful first to identify the basic structure of DNA and how it is stored inside the cell. DNA is located inside the cells of all species. Eukaryotic cells are cells that have a true nucleus; a membrane bound organelle in which the DNA is contained. The nucleus is the “control center” that directs all cellular activities. The entire cell is bound by a cell membrane (also called a plasma membrane), and the nucleus is surrounded by a double nuclear membrane (called a nuclear envelope). These barriers are both made up of two layers of fatty, oily compounds called phospholipids. The DNA of eukaryotic cells, if the chromosomes were stretched out and laid end-to-end, is about 100,000 times (!) the diameter of the cells themselves. However, DNA only takes up about 10% of the total volume of the cell. This is because DNA is highly folded and packaged into structures called chromosomes within the nucleus. A chromosome is a bundle of DNA tightly wound around protein molecules. DNA is not visible to the eye unless it is amassed in large quantity by extraction from a considerable number of cells. 14478002794000047117001790700Figure 1 00Figure 1 When chromosomal DNA is unfolded and the proteins coating it are removed, the DNA structure is exposed as a twisted ladder called a double helix. The sides of the ladder form the DNA backbone with alternated sugar and phosphate molecules. The rungs of the ladder are comprised of pairs of nitrogenous bases [adenine (A) with thymine (T) and cytosine (C) with guanine (G)] joined by hydrogen bonds (see Figure 1). Although the structure of DNA is well known and clearly defined, even the most powerful microscopes cannot visualize the DNA double helix of chromosomes. All living things are dependent on DNA, and the structure of DNA is consistent among all species. However, the particular sequence of nucleotide bases within DNA molecules differs between organisms and from gene-to-gene to create explicit “blueprints” unique to each individual. This sequence of base pairs is what makes an organism an oak tree instead of a blue jay, etc. The DNA of a animal or plant cell is located within the cell’s nucleus which is located in the cytoplasm of the cell. The nucleus and the organelles in the cytoplasm are enclosed by phospholipids that protect and separate the cell from its surrounding environment. Therefore in order to extract the DNA from the cells, the cell wall (if using a plant cell), cell membranes and nuclear membranes must first be broken. The process of breaking open a cell is called “cell lysis”. Physical actions such as mashing, blending, or crushing the cells cause the cell walls of the berry to burst. The cell membranes and nuclear membranes may be disrupted with a detergent-based extraction buffer. Just as a dishwashing buffer dissolves oils and fats (lipids) to cleanse a pan, a detergent buffer dissolves the phospholipid bilayer of cell membranes. Once the cell wall and cell membranes are ruptured, the cell contents flow out, creating a soup-like mixture of DNA, cell wall fragments, dissolved membranes, cellular proteins and other contents. This “soup” is called the “lysate” or “cell extract”. The DNA molecules can then be isolated away from the cellular debris in the lysate. For this purpose, the detergent-based extraction buffer also includes salt. The salt causes some of the cellular debris in the soup to precipitate out of solution while the DNA remains dissolved. This means the cell debris become suspended particles that can be seen. The cell extract is then filtered through layers of cheesecloth. The cheesecloth traps the precipitated cell debris while the soluble DNA passes through. DNA is soluble within the aqueous cellular environment and in the presence of the extraction buffer, but is insoluble in alcohol (such as ethanol and isopropanol). Applying a layer of ethanol on top of the filtered lysate causes the DNA to “precipitate”, forming a translucent cloud of fine, stringy fibers at the point where the alcohol and cell extract meet. Cold ethanol works best to precipitate DNA to the fullest. DNA extracted from multiple cells is visible by eye and can be wound onto a wooden stick in a process known as “spooling” the DNA. Strawberry cells are excellent sources of DNA for extraction. They are multicellular and contain eight copies of each of their seven chromosomes in each cell. As a result, just one berry will yield enough DNA to be easily seen and spooled. Strawberries are also a soft fruit, which makes them easy to mash. Mashing the berries breaks down the strawberry tissue, releases individual cells, separates the seeds from the cells, and breaks the cell walls. In addition, ripe strawberries produce pectinase and cellulose-enzymes that contribute to the breakdown of cell walls. Exercise I – Extracting DNA MATERIALS NEEDED You will need to prepare beforehand to complete this activity. To do so, obtain the following materials: One fresh or frozen strawberry with the green sepal removed. Extraction buffer: Combine 1 teaspoon (5 ml) liquid dish detergent (Dawn or Palmolive works the best) with 6 tablespoons of water and 1/2 teaspoon table salt One ounce of isopropyl alcohol (rubbing alcohol) stored in freezer for at least 4 hours making it ice cold. 1 Small zip-lock bag 1 Piece of cheese cloth that will fit into a small funnel or strainer 1 Kitchen funnel or fine-mesh strainer 1 Eyedropper 1 Clear small tube with a diameter of a thick highlighter pen. (Note: This may be the most difficult item to find. A test tube or a small bottle not bigger than a fat highlighter is ideal…) 1 Small wooden stick 1 Cell phone or digital camera to record the steps and final product. PROCEDURE 1. Obtain one fresh or one frozen and thawed strawberry. If you are using a fresh strawberry, remove the green sepals (tops) from the berry. 2. Place the strawberry in a re-sealable zip-lock plastic bag. 3. Close the bag slowly, pushing all of the air out of the bag as you seal it. 4. Being careful not to break the bag, thoroughly mash the strawberry with your hands for two minutes to obtain a fine pulp. 5. Measure out 2 teaspoons (10ml) of the extraction buffer. Then carefully pour the buffer into the bag with the mashed strawberry. Reseal the bag. 6. Mash the strawberry and buffer together for 1 minute. Try to squish out all the lumps of berry. **RECORD A PICTURE OF YOUR EXTRACT IN THE BAG** 6. Place a funnel on top of a small juice glass. Take the cheese cloth and place it into the funnel or strainer to form a filter. The cheesecloth will overlap the edge of the funnel. 7. Carefully pour the strawberry mixture into the funnel, filtering the contents through the cheesecloth and into the glass. 8. Carefully pour 1/2 to 3/4 of a teaspoon of the filtered contents from the juice glass into your clean test tube or small bottle. If your tube does not have graduations on the side use an eye dropper to place the red filtered contents in the tube. 40-45 drops will equal 1/2 to 3/4 of a teaspoon. **RECORD A PICTURE OF YOUR EXTRACT IN THE BOTTLE/TUBE** 9. Hold your tube AT A 30-DEGREE ANGLE. Using a clean eye dropper (BE SURE TO CLEAN THE EYE DROPPER IF YOU USED IT PREVIOUSLY TO ADD THE LYSATE TO YOUR TUBE!), carefully and SLOWLY add 75 drops of the ice cold (very important!) ethanol or isopropyl alcohol DOWN THE SIDE OF THE TUBE. You should have two distinct layers. Do not add the drops right on the red layer, drop them on the side of the tube so they run down the side of the tube and layer on top of the red strawberry extract. CAUTION: DO NOT MIX THE STRAWBERRY EXTRACT AND THE ETHANOL. YOU NEED TO HAVE TWO DISTINCT LAYERS IN THE TUBE. **RECORD A PICTURE OF YOUR LAYERS IN THE BOTTLE/TUBE** 10. Watch closely as translucent strands of DNA begin to clump together where the ethanol layer meets the strawberry extract layer. Tiny bubbles in the ethanol layer will appear where the DNA precipitates. This may take a few minutes. 26670003048000011. Slowly and carefully rotate the wooden stick in the ethanol directly above the extract layer to wind (or “spool”) the DNA. CONGRATULATIONS! You have purified DNA from a strawberry! Remove the wooden stick from the tube and observe the DNA **RECORD A PICTURE OF YOUR DNA SPOOL ON THE WOODEN STICK** 12. To complete this assignment, answers to the questions below. Exercise II – Recording Observations Please insert the (4) pictures you were asked to record in the appropriate space below: **RECORD A PICTURE OF YOUR EXTRACT IN THE BAG** **RECORD A PICTURE OF YOUR EXTRACT IN THE BOTTLE/TUBE** **RECORD A PICTURE OF YOUR LAYERS IN THE BOTTLE/TUBE** **RECORD A PICTURE OF YOUR DNA SPOOL ON THE WOODEN STICK** Exercise III – Lab Questions Please enter your responses to the questions below to submit along with your lab observations: Did you see any DNA strands? If so, what did they look like? If not, please explain why you think it did not work. Click here to enter text. Describe the structure (in detail) and the general function of DNA. Click here to enter text. How does mashing the strawberries help to extract DNA? Why? Click here to enter text. Why is salt added to the detergent based extraction buffer? Click here to enter text. Do you think the DNA extracted from animal cells would look the same as DNA from plant cells? What are the differences/similarities between our DNA and plant DNA? Please justify your answers with scientific reasoning and concepts. Click here to enter text. Give three examples of ways research scientists use extracted DNA from a plant or an animal. Click here to enter text.