DNA Extraction of a Musa acuminata

A number of steps are required to isolate DNA from cellular contents. Describe what happens at each step, and why it acts to separate the parts of the cell. /6 There are three specific steps required to isolate DNA from its cellular contents. The steps used to remove and expose DNA from its cell are: breaking down the food type you are using by crushing it, for example a banana or strawberries, exposing the substance to a sodium chloride (NaCl) solution, subjecting the product to detergent solution (dH2O), filtering the solution and lastly, the addition of ethanol. When beginning with a solid substance, such as a banana, crushing the substance allows for breaking down the cell membrane and cell wall, if using a plant cell. This ultimately, removes the contents of the cell from the deoxyribonucleic acid (DNA) and further exposes it for the detergent solution. After this step, one would use NaCl solution to stabilize the DNA, since we know that it is negatively charged, due to the phosphate functional group. The way this occurs is when NaCl is added to the solution the molecule dissolves and forms ions.

As a result, the positively charged ions neutralize the negative charge on the DNA molecule (“Discovering DNA” 20 Feb. 2014). The technique used to separate the DNA is by adding a detergent liquid to the solution, which allows for the cell membrane to break down. This step specifically emulsifies the phospholipids and proteins that make up the cell membrane (“Discovering DNA” 20 Feb. 2014), in order to further expose the DNA. Then in order to separate the DNA from the proteins and lipids one must use a filter paper with an appropriate pore size so that the flow rate of the solution is effective. Once the flow rate is minimal, the excess in the filter, consisting of cell debris and banana residue, can be disposed and then the filtrate is focused upon. With the specific amount of filtrate in a gas tube, one must add ice-cold 95% ethanol, which will float due to its lower density. Theoretically, the reason for the cold temperature is so that it can reduce the activity of enzymes found in the cytoplasm that can potentially harm DNA, since there is no nuclear membrane (“How to Extract DNA from Anything Living” 20 Feb. 2014). Also, the alcohol precipitates the DNA out of the filtrate to make it rise and visible in the clear solution (“Extracting DNA from Living Things” 20 Feb. 2014). Overall, these are the main steps needed to isolate DNA and each step above explains the mechanics behind it.

Do you think the process or results would be different if you were to use a vegetable, animal cells, or a different fruit? Explain. /2 The processes and results of this extraction will vary, even though structurally the DNA molecule is relatively the same, depending on three main ideas, one of which being the fact that fruits and vegetables vary in their pH levels. For example, an orange’s acidity affects the DNA by altering it, which if one further examines the DNA, the analysis will be different. For this reason one may pick a cell, that is more alkaline as to not effect the DNA. Also, another key difference between an animal cell and plant cell is the fact that in order to reach the animal cells one must pass through a cell membrane, while a plant cell has two barriers: a cell wall and cell membrane. Lastly, “the amount of DNA you will get will depend on the ratio of DNA to cell volume rather than the number of chromosomes in your material” (“Extracting DNA from Living Things.” 20 Feb. 2014).

This is very true because one may have an effective process, but after passing through all these layers the cell may not have much DNA, reducing your percent yield. Overall, these three factors affect the result of using different cells. What property of DNA allows it to be “coiled” around a glass stirring rod once precipitated? /2 The property of DNA that allows for it to coil around the glass rod is due to two reasons. Firstly, DNA is constantly trying to coil itself back into its original position, which is a supercoiled polymer. So when the DNA is exposed to a stirring rod in a circular motion the DNA tries to coil itself to the rod. Secondly, another property of DNA, which is the fact that it is polar, causes it to attract to the polar glass rod. These two properties working together allow the DNA to coil around the stirring rod. Conclude and Communicate

Based on your procedure and results, how is DNA isolated from plant or animal cells? /4 When looking at DNA extraction, the key steps for isolating DNA from a plant cell or animal cell is to pass through the protective barriers of each cell – whether it is a cell wall and cell membrane or only the cell membrane for an animal cell. In order to extract DNA from a banana, the first step required is to place half of it in a small resealable plastic bag without air bubbles, and then using your fingers mash the banana until no visible chunks. Next, using a graduated cylinder measure 10 mL of the 0.1% NaCl solution, and add it to the bag – mix thoroughly. Then the next step is with a graduated cylinder measure 3 mL of detergent solution (dH2O), add it to the bag and mix gently, so as not to form bubbles. After this, set up a retort stand with the gas tube using a retort clamp, strain the banana mixture through a filter paper, and collect the filtrate in a gas tube for approximately 20 to 30 minutes. Once the flow rate of the solution is minimal remove the banana residue and transfer 5mL of the filtrate into a separate dry gas tube.

Lastly, using another graduated cylinder measure 8 mL of room temperature ethanol, tilt the test tube and gently add the ethanol by pouring it down the side, so that the filtrate at the bottom of the tube is not disturbed. The result one should see is the alcohol layer floating on the banana filtrate. Almost instantly the DNA should begin to precipitate in the clear ethanol (the top layer); though let the gas tube sit for 2-3 minutes. (Carter-Edwards, et al. 230). If one sees a visibly dense white substance, the extraction is successful. This process works best for plant cells, but there can be many ways to improve the results of this experiment (discussed in question 6). As you may realize there are many steps required to isolate DNA in a plant cell due to its complex protective layers and as a result, since animal cells have a single barrier, cell membrane, it is easier to extract DNA from most animal cells without the use of enzymes. Describe the appearance of the DNA collected. /1 The DNA’s appearance should appear to be a dense white stringy substance that consists of many fibers of DNA. They overall structure can be like a cotton ball or it can be spread throughout the gas test tube depending on the size of the gas tube and how well you DNA is extracted. Extend

What other steps could be taken in this procedure to better purify your DNA sample? /10 The other steps that can be taken for this procedure are centrifugation, incubation, DNA washing and the use of enzymes; all these steps can help to further refine the DNA sample. Firstly, the use of a centrifuge can be very effective to separate the substances, in the solution of detergent, NaCl and the specific cell being used, by spinning it at high speeds. This would cause the heavier proteins and lipids to settle at the bottom becoming the pellet, while the DNA would remain at the top – supernatant. Then by removing just the supernatant, which holds the DNA, one would then add the ethanol and repeat the process of centrifugation, but this time the pellet would be the DNA as it has precipitated. This technique is very effective as it allows small amounts of DNA to be extracted. The second possible method that could be used before adding the NaCl solution is incubation, where one would excite the molecules in the cell. This would catalyze the process of breaking down the phospholipid membrane, and denature parts of the protein in the membrane (“Wheat Germ DNA Extraction” 20 Feb. 2014). Ultimately, this would help break down the cell membrane effectively and further expose the cell.

The third technique used to ensure there is no contamination and impurities is the washing of the DNA pellets that are collected after the first centrifugation with 70% ethanol, instead of 95% ethanol. The reason this is effective is that it removes the salts from the DNA molecule without affecting it. This occurs due to the property of salt being soluble in 70 % ethanol, but as one increases the concentration, the solubility of the DNA decreases allowing it to precipitate (“Wheat Germ DNA Extraction” 20 Feb. 2014). The final step that one might decide to take is enzymes, which would target specific substances in the solution. For example, RNase, which would break down the RNA in the solution, since this molecule tends to intertwine itself with the coiled DNA. Also, the use of meat tenderizer, which is a household ingredient used to season meat, contains papain – protease enzymes extracted from papaya – which would help break down protein and histone structures that allows for the DNA to uncoil (“How to Extract DNA from Anything Living” 20 Feb. 2014). Also, this protease helps to further emulsify the membrane that is also made of proteins (“Wheat Germ DNA Extraction” 20 Feb. 2014). Overall, I believe that these steps can be effective to further purify the DNA sample and allow for better results.

Works Cited

Carter-Edwards, Trent, et al. Biology 12. Canada: McGraw-Hill Ryerson Limited, 2011. Print.
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“Extracting DNA from Living Things.” Nuffield Foundation. N.p., 2011. Web. 20 Feb. 2014.
“Frequently Asked Questions.” Genetic Science Learning Center. N.p., n.d. Web. 20 Feb. 2014.
“How to Extract DNA from Anything Living.” Genetic Science Learning Center. N.p., n.d. Web. 20 Feb. 2014.
Jansons, I. “How to Extract DNA from a Banana.” Queens University Resources. N.p., n.d. Web. 20 Feb. 2014.
Josephs, Molly. “Finding the DNA in a Banana.” Scientific American. N.p., 2011. Web. 20 Feb. 2014.
Weibel, Jnaneshwar T.. “DNA Extraction from Plant and Animal Cells.” California State Science Fair. N.p., n.d. Web. 20 Feb. 2014.
“Wheat Germ DNA Extraction.” Virtual Lab Book 5th Edition. N.p., n.d. 2000. Web. 20 Feb. 2014.