The Isolation Of DNA From Onion Cells
DNA (deoxyribonucleic acid) can be isolated from material by homogenizing, deproteinizing, and precipitating it from said material. Procedure: The first step in DNA isolation is homogenization, which involves breaking down and removing cell walls and membranes. I prepared a medium consisting of 50.0 g of sodium dodecyl sulfate (SDS) 8.77 g of sodium chloride (NaCl), 4.41 g of sodium citrate, 0.292 g of ethylenediamine tetraacetic acid (EDTA), and enough distilled water to make 1 liter of solution. The detergent-like action of SDS helps to dissolve cell membranes and denature proteins. The NaCl/sodium citrate buffer stabilizes the DNA by forming a Na+ shell around the negatively charged phosphates of the DNA. The citrate inactivates the DNAse that would otherwise break down the DNA. I diced the six onions and weighed out 50.0 g into 250 ml beakers. I then added 100 ml of the homogenizing medium to each sample and incubated them into a 60° C water bath for 15 minutes. The heat softens onion tissue, allows the medium to penetrate, denatures many enzymes and proteins, and helps to prevent denaturing of DNA. I cooled each sample in a 15° ice bath to prevent denaturing of DNA. I blended each preparation for 45 seconds at low speed and 30 seconds at high speed. Blending breaks open cells and releases their contents. I cooled these homogenates in an ice bath for 20 minutes and filtered each homogenate through quadruple-layered cheesecloth. These preparations were stored at room temperature until further experimentation continued. The second step in DNA isolation is deproteinization. This further purifies the DNA by removing proteins left from the homogenization step. First, I poured 50 ml of each homogenate into 50 ml flasks and added 2 ml of chloroform to each homogenate. After swirling the mixture, the chloroform forms a layer (phase) below the homogenate and denatured proteins collect in-between the other two phases. I poured the homogenate into another 50 ml flask and collected waste chloroform and proteins in a glass jar. I repeated this chloroform cycle for each homogenate 4 more times, for a total of 30 chloroform cycles. For the last cycle of each homogenate, I was careful to leave behind all of the chloroform layer, because the chloroform would contaminate the homogenate. I stored the purified homogenate at room temperature. The last step in DNA isolation is the actual precipitation of the DNA. I cooled each deproteinized homogenates in a 15° ice bath and then added 80 ml of -10° ethanol to each homogenate. A layer of DNA formed between the ethanol and homogenate, and I wound up the DNA onto glass rods, placed the rods in test tubes filled with ethanol, and stored them at 8° in a refrigerator. I had planned not only to prove that I could extract DNA from different species of onions, but also to prove that DNA from each species of onion would be different. I hoped to make a "bar-code" of each type of onion species by separating its deoxyribonucleic acid into a pattern of bands through a technique called gel electrophoresis. The main steps in electrophoresis are: 1) resuspend the DNA, 2) add the buffer and enzymes, 3) make the gel, 4) load and run the gel, 5) stain, view, and photograph the gel. To resuspend the DNA, I tore off a small piece of DNA using dissecting forceps and added approximately 40 m l of distilled water. I extracted about 10 m l of my DNA solution and added approximately 1 m l of two restriction enzymes, Bam Hl and Eco Rl, along with 1 m l of corresponding reaction buffer (each substance should be at -20° ). I allowed approximately 30 minutes for my restriction enzyme to work, and then added 1 m l of Final Carolina Blu, an alternative to the more famous carcinogen ethidium bromide. These samples were now ready to be placed into the finished gel. My gel was made according to directions received within the package, by adding 0.24 gm of Agarose along with 0.6 ml of buffer and 29.4 ml of distilled water for each cube. I used two cubes in my experiment, bringing my total up to 0.48 gm of Agarose along with 1.2 ml of buffer and 58.8 ml of distilled water. This mixture was heated in the microwave until clear, and then allowed to cool to 55° C while swirling the solution. I carefully removed my gel, and slid each cube into the proper electrophoresis chamber. I filled each chamber with enough buffer to cover the indents of holes created by the comb placed within the gel. At this point in time my chamber was hooked up and prepared to run a cycle. Each sample was loaded into every other hole (since there were 12 holes and only 6 samples). The chamber was placed within an electric field, and each sample was allowed to run for 1 hours time. At this point in time there was no banding showing, and I became worried as to the final results. The test was performed to the time of 3 hours, and each gel was stained and washed (destained). I found at this time that my gel showed no banding whatsoever, and this is also the time when I noticed my buffer and separated and moved in particles towards the positive (+) end. There are several explanations as to why this part of experiment did not work, as described in the next paragraph. One of the main reasons I feel the second half of my experiment was unsuccessful was the improper tools used. Because I was unable to locate a micropipette at the time, transfer pippetes were used. These instruments are not precise, and a significant amount of my sample was wasted in the process. Because of this I feel an insufficient amount of DNA was transferred into each well, therefore the tiny strands that did run the process did not show up. Another possibility is that the buffer was improper, as shown by the fact that it separated and moved towards the positive end. Because electrophoresis requires a large amount of buffer, capsules were used that dissolved in water (a cheap alternative). After the experiment was completed, I noticed that these capsules had expired. This is another factor that may have contributed to the failed experiment. However, I accidentally proved that these Hydrion buffer molecules have a negative charge, which is why they traveled to the positive electrode. I attempted to reconstruct my experiment using better tools and new materials. At this point in my experiment I used much better equipment (a micropipette) and a buffer that came highly recommended by Edvotec, Tris-acetate-EDTA (20 mM tris, 6 mM sodium acetate, 1 mM of disodium ethylenediamine tetraacetic acid). This 50x buffer was diluted by adding 6 ml to 294 ml of distilled water, making my total buffer count 300 ml. I ran my gel for the time of 18 hours (I had originally assumed I was dealing with a 25v machine, when in reality its output was about 8 volts). Both gels were stained for 30 min. and destained for 45 min. At this point in time, 2 sets of bands showed on the yellow onion gel, and 3 bands were showing on the red onion gel. The first set of bands showing on the yellow onion gel were very dark, and located 3.15 cm from the well from which they originated. The second, much fainter set of bars were located 4.45 cm from the well from which they originated. In each yellow onion tested, the pattern remained the same. The red onion samples produced a three-bar pattern. The first set of bars were once again the darkest (about 2.80 cm from well). The second bar set was lighter, and located about 3.95 cm from the well. The third and final set was the lightest, approximately 5.30 cm from the well. By repeating this experiment, I was able to prove that the DNA pattern for each onion species was different, and members within that species share that pattern. Conclusion: DNA can be isolated from its surrounding matter through a process involving homogenization, deproteinization, and the eventual separation of DNA. "Hydrion" molecules have a negative charge, as accidentally demonstrated by their attraction to the positive electrode in an electrical field. Each species of onion plant has its own DNA pattern, and members within each species share a pattern.