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Isolation and Characterization of Onion DNA

The experiment was about the isolation and characterization of DNA. The DNA was isolated from the onion. The mass of the isolated DNA was 15.11 g. The purity of isolated DNA was estimated by calculating the ratio based from the absorbance at 260nm and 280nm resulted to 0.671 meaning more protein was absorbed. Meanwhile in denaturation of DNA, the initial absorbance at 260 nm was 1.304 higher than the absorbance at 260 nm after heating which was 1.095.
INTRODUCTION Deoxyribonucleic acid (DNA) is the genetic material in humans and all other organisms. DNA isolation is the removal of DNA from the cell which it normally resides. Isolation is the removal of DNA from the cell in which it normally inhabits. (1)
Onions are used since it contains little amount of starch which allows the DNA to be more visible. The filtrate is made up of onions treated with salt, distilled water and detergent collectively called as lysis solution. DNA purification is done by enzymatic degradation of contaminating proteins with ethanol. A spectrophotometer is used in determining the concentration and purity of the proteins. (2)
MATERIALS AND METHODS Isolation of DNA from Onion
The peeled onion bulb was chopped and measured homogenized. The sample was placed in a blender added with an ice-cold lysis solution then for 45 seconds at low speed. Meanwhile, the lysis solution used was prepared beforehand by mixing 5.00 ml of liquid detergent, 5.00 ml of 0.500M EDTA, 10.0 ml of 50% Na Cl solution, and 80 ml of distilled water and placed in an ice bath. After homogenizing, the sample was filtered through the cheesecloth and the collected filtrate was placed in a 250-ml beaker. A 10.0 ml of 5% pepsin solution was added to the filtrate and placed on an ice bath for 10 minutes with occasional stirring. Ice cold 30.0 ml of 95% ethanol was pipette to the side of the beaker containing the sample and stand for 10 minutes on ice bath. Once the DNA precipitates appeared at the interface of the solution, the DNA was already ready for isolation. The spooled DNA was transferred immediately to a pre-weighed 100-ml beaker to determine the mass and percent yield of the sample. The isolated DNA was added with 10.0 ml of 95% ethanol then covered with aluminum foil and refrigerated in preparation for the next laboratory procedure.
Characterization of DNA
Little amount of DNA sample was placed in a test tube added with 1.00 ml of 20% TCA followed by heating the sample for 10 minutes in water bath with 1.00 ml distilled water. A 2.00 ml of diphenylamine solution was added then heat again in a water bath for 10 minutes. The color change was observed and the absorbance of the sample from 400 nm to 700 nm was scanned to determine the wavelength of maximum absorption. Mean while, little amount of the DNA sample was placed in a separate test tube filled with 5.00 ml distilled water and scanned to read the absorbance at 260 nm then at 280 nm. After determining the A260/A280 value, the sample was heated to boil for 5 minutes and read the absorbance adain at 260 nm.
RESULTS AND DISCUSSIONS The mass of the raw sample gathered from onion is 30.4 g. After homogenization and adding of pepsin solution and ethanol, DNA precipitates were became visible and transferred to another beaker. The isolated DNA measures 23 g.
The calculated percentage yield was quite high. However, still some sources of error was done while conducting the experiment, the sample with DNA precipitates was disturbed while transferring the DNA. The accumulated DNA precipitates is enough for the next procedure which is characterization.
Heat denaturation of DNA, causes the double helix structure to unwind and form single stranded DNA. Thus, the bases unstacked and can absorb more light causing an increase after denaturation. But based on the results gathered, the initial absorbance at 260 nm was 1.304 then was decreased after heating which was 1.095. The calculated percent increase in absorbance was 8%. This error is maybe, due to the heating process. The DNA acquired was quite greater and was not totally heated afterwards causing double helix structure not to unwind and form a single stranded DNA.
The filtrate gathered from this experiment was made of onions and lysis solution. Onion was used in this study due to low starch content, allowing the DNA to be more visible considering the onion as one of the best source of DNA. (4)
The used of lysis solution was to separate the DNA from extra cell components and to keep the location in which the DNA will not be tainted. The NaCL provides NA ions that will obstruct the negative charge as of phosphate ends of DNA. Permitting these ends to come nearer so they can precipitate out of a cold solution. The detergent causes the breaking down of the cell membrane by emulsifying the cell proteins and lipids. Also, disrupting the polar connections that collectively holds the cell membrane. The complexes formed with these lipids and proteins causes the precipitate out of solution. Meanwhile, the purpose of EDTA is to chelates metal ions. (5) A Pepsin solution was used for purification via enzymatic degradation.
DNA is polar due to its extremely charged phosphate backbone which makes it soluble in water. Thus DNA is insoluble in ice cold ethanol, as a result when the cold ethanol was added, it causes stable ionic bonds to form and precipitate the DNA.
Heating the sample is the one responsible for the formation of the observed color of DNA with diphenylamine. When the DNA is heated with acid, the 2-deoxyribose is converted to w-hydroxylaevulinic aldehyde, which reacts with the compound diphenylamine. Through this, a blue-colored compound supposed to produce. In our sample the color observed was green possibly because of the DNA concentration.
The ratio of absorptions at 260 nm vs 280 nm is frequently used to evaluate DNA contamination of protein solutions. The nucleic acids, DNA and RNA, absorbs at 260 nm and proteins absorb at 280 nm. Based on the results, the rate ratio of absorptions at 260 nm vs 280 nm is 0.671. Since proteins absorb light at 280 nm, the ratio is low meaning there is a lot of protein absorbed at 280nm.

Length of Pelvetia Canaliculata on Upper Shore Zone

The aim of this study was to compare the length of Pelvetia canaliculata on the upper shore zone of both wave-sheltered and wave-exposed shores. The hypothesis was that the Pelvetia fronds growing on the upper shore zone of a wave-sheltered rocky shore will be significantly longer compared with fronds growing on the upper shore zone of a wave-exposed rocky shore.
The lengths of 450 fronds in total were sampled using systematic continuous horizontal belt transect sampling method at a wave- exposed and wave-sheltered shores on the Pembrokeshire coast. The results showed that there was a significant difference in the lengths of the seaweed with longer fronds being found in the wave-sheltered rocky shore. This is happening because there is less wave exposure and so fronds are less likely to be broken off at the tips and hence they will be longer.
INTRODUCTION Rocky shores are areas of bedrock exposed between the extreme high and extreme low tide levels on the seashore. The ecosystem is complex, as it is an interaction between terrestrial and aquatic systems. Plants and animals are distributed on the shore in horizontal zones that relate to the tolerance of the species to either exposure to air or submergence in water during the tidal cycle. This zonation can be very clear and abrupt. Using this fact, I have clearly identified the area Pelvetia canaliculata is found; the upper shore. [1] ­I have researched this zone in more depth to be able to understand the conditions, the problems and the different variables that can affect Pelvetia canaliculata and the adaptations it developed to survive and thrive.
As the tide level drops on the upper shore, the seaweed will be exposed to air and desiccation (drying out) occurs as a result. Because the tide rises up and down twice a day organisms at the top of the shore get much less water than ones at the bottom. Across a year, the top of the upper zone gets covered by the sea for less than 1% of the year while The bottom of it for about 20% of the year. This is very short time to obtain nutrients from the water for photosynthesis, and can therefore slow growth rate. However, this is not the only problem as the water filters off some of the wavelengths of light and reduce its intensity and so lowering photosynthesis rate. In addition, the water is the main medium where dispersal of spores happens; spending less time in the water means low productivity. [2]
Species on the Upper shore get subjected to a wide variation in temperature. Immersion in water buffers against temperature change due to the high specific heat capacity of water. Upper shore species will have to tolerate the greatest variation in temperature whilst it has least effect in the lower shore. High temperatures will increase the risk of desiccation and increases salinity in pools. [3]
The other major physical factor that controls what can live on a shore is wave action. Exposed shores have a lot of wave action and sheltered shores have little. Seaweeds find the drier, brighter, wave exposed environment very difficult to cope with.
Sheltered shores [4]
Exposed shores [4]
“Usually face away from the open sea and the prevailing wind. This means they generally have smaller waves than exposed shores which face out into the open sea and the prevailing wind.
Sheltered shores are usually on north to north easterly facing shores. North facing sheltered shores get less sunlight than exposed ones, and are less susceptible to desiccation and in general are more hospitable places for inter-tidal organisms.”
“Usually face into the open sea and the prevailing wind. This means they generally have bigger waves than sheltered shores.
Exposed shores are usually on south to south westerly facing shores. South to south westerly facing exposed shores get more sunlight than sheltered ones, are more susceptible to desiccation and in general are not hospitable places for most inter-tidal organisms.”
Now that I explained the features of the upper shore zone and the exposed and sheltered shores I will describe the features and the different adaptations that enabled Pelvetia canaliculata to live in such a habitat and constantly changing environment.
Taxon English equivalent or translation [3]
Phylum Chromophycota /Brown seaweeds e.g. kelps