The intent of the experiment was to see firsthand the isolation of DNA organize a works tissue without destructing its construction and sequence. A white onion was used for the experiment. After several procedures. Deoxyribonucleic acid isolate was the seeable consequence. Different chemical trials were performed on the Deoxyribonucleic acid isolate. viz. : Dische Test. Murexide Test. Wheeler-Johnson trial and Test for Phosphate. Visible consequences were so noted.
Deoxyribonucleic acid ( deoxyribonucleic acid ) was discovered in the late 1800s. but its function as the stuff of heredity was non elucidated for 50 old ages after that.
It occupies a cardinal and critical function in the cell as the familial information in which all the information required to double and keep the being.
Today. Scientists can analyse Deoxyribonucleic acid from infinitesimal samples of blood. hair roots and spit. but before they can analyse DNA. scientists must be able to pull out it. The procedure of DNA extraction is the first measure for many research lab processs.
Scientists must be able to insulate the Deoxyribonucleic acid from the other unwanted substances of the cell gently plenty so that the DNA is non broken up.
For this experiment. an onion was used because of its low amylum content. which allows the Deoxyribonucleic acid to be more clearly seen. The salt shields the negative phosphate terminals of DNA. which allows these terminals to come closer so that the Deoxyribonucleic acid can precipitate out of a cold intoxicant solution. The homogenising solution causes the cell membrane to interrupt down by fade outing the lipoids and proteins of the cell. which disturbs the bonds that hold the cell membrane together. The homogenising solution so forms composites with the lipoids and proteins doing them to precipitate out of the solution.
After the isolation of the Deoxyribonucleic acid from the solution. different chemical trials were so performed on the isolate. which were: The Dische Test. Murexide Test. Wheeler-Johnson Test and Test for Phosphate.
The Dische trial is a trial done to place DNA. It is the reaction between Dische reagent & A ; 2-deoxypentose outputs a bluish coloured solution.
The Murexide trial a trial in which the intervention of a substance. normally urine. with azotic acid and ammonium hydroxide indicates the presence of uric acid by formation of murexide. Positive consequences form this trial yields Purines form Yellow to Red-Violet.
The Wheeler-Johnson Test is a qualitative trial for the pyrimidine bases C and U. which produces a green colour when the sample is treated with bromine H2O. The add-on of Ba hydrated oxide will turn the liquid purple.
The Test for phosphate is used to observe phosphate in the Deoxyribonucleic acid. positive reaction outputs xanthous precipitate.
RESULTS AND DISCUSSION
The undermentioned processs were done in order to insulate the Deoxyribonucleic acid from the onion. First. the onions were minced and so weighed to 25g. The onions were minced because by softening the onions. it increase its surface country that helps do the membrane at the surface easier to fade out. and besides allows for more efficient soaking up of heat and solutions.
Next it was added to a 50ml homogenising solution. this was done because the homogenizing medium. made of 5 % SDS. 0. 15M NaCl. 0. 15 M Na citrate and 0. 001 M EDTA. was similar to a detergent. It helped interrupt up the phospholipid bilayer of the cells’ plasma membranes and atomic envelopes. When in SDS. the proteins and lipoids of the membrane. but particularly the lipoids. are broken down because the SDS causes the bonds keeping the membrane together to interrupt. The lipoids. which are repelled by the SDS. separate and interrupt up the membrane. This is why detergent is used to take a lubricating oil discoloration ; lubricating oil is a lipid. The EDTA helped weaken the membrane every bit good. doing the Deoxyribonucleic acid that was in the karyon now available for pull outing.
Then the solution together with the onion was heated at 60 grades. this method was used to rush the dislocation procedure by stimulating the molecules. and besides. hopefully help to fade out the phospholipid bilayer by destructing the proteins and interrupting the bonds that hold the phospholipids in topographic point. By exciting the molecules. the heat might do the fluid bilayer to interrupt and go a gas-like substance. no longer functioning as a barrier for the Deoxyribonucleic acid. The heat softens the membrane as a whole. This full measure. homogenation. is used to liberate the Deoxyribonucleic acid from its protective barriers.
Then petroleum papain was added. because Deproteinization which involves adding a peptidase enzyme Papain will denature the proteins cleaving to the Deoxyribonucleic acid doing the molecule flexible and easy to spool. Then the solution was so heated once more to 60 grades for 10 proceedingss with occasional stirring.
Then it was placed in an ice bath. this is because by chilling the solution. it will assist forestall denaturation. which might destruct the Deoxyribonucleic acid if it was exposed to protract warming. Twirling was done in order non to “shock” the Deoxyribonucleic acid from the sudden alteration in temperature.
After seting it in an ice bath. the solution was so placed in a liquidizer and was blended for 45 seconds. after. This was done because the process besides frees the Deoxyribonucleic acid from another protective barrier. the midst cell wall that all works cells have. The blending should do many of the cell walls. incorporating cellulose. to interrupt. and hence leave the Deoxyribonucleic acid in the cytol. and no protective midst cellulose wall barricading the cytol from its external environment. We did non necessitate to make this to the bacterium because it does non hold a cell wall.
The blended solution was so filtered through 4 beds of cheesecloth go forthing the froth untouched. This is because it leaves behind thicker stuffs. including any parts of the staying onion itself. The liquid that is filtered through contains the DNA. no longer held behind a plasma or atomic membrane. and ready to pull out. All waste non needed for the experiment is filtered out. such as cellulose of the cell walls. pectins. and extra tissues like the tegument sclerenchyma cells of the onion.
Then the volume of the isolate was measured and was so transferred onto a 250mL beaker. and was so placed in an ice bath. so beaker was so tilted to 45 grades and 95 % ice-cold ETOH was added. The analysis of this was that the DNA is polar. but the reaction with the ethyl alcohol makes the DNA nonpolar. and hence immune to the homogenising medium that it is in. The Deoxyribonucleic acid forms its ain bed. a precipitate between the liquid ethyl alcohol and liquid homogenate. Besides. the Deoxyribonucleic acid is the lone constituent of the solution that is non soluble in the ethyl alcohol. Therefore. it becomes its ain clear bed on top of the igniter ethyl alcohol. but free of the soluble homogenizing medium. However. the reaction above. with the Deoxyribonucleic acid precipitating to the top of the homogenate because it becomes nonionic and staying under the ethyl alcohol because it is non soluble merely happens at the top of the solution where the Deoxyribonucleic acid is exposed to the ethyl alcohol and does non travel far to precipitate out. Therefore. the top Deoxyribonucleic acid remains between the two beds.
Last. the Deoxyribonucleic acid was spooled and placed in a foil. so it was air-dried for rather sometime. so was prepared for the different coloured reactions.
Dische Test ( Standard: D-Ribose Solution )
( Standard: Guanine )
( Standard: Cytosine )
Trial for Phosphate
( Standard: Conc H3PO4 )
Deoxyribonucleic acid Isolate
Faint bluish solution formed
Yellow residue formed
White solution formed ; ruddy litmus paper turned bluish
Clear. colorless liquid formed
Dark bluish solution formed
Orange residue formed
Violet solution formed ; ruddy litmus paper turned bluish
Faint xanthous precipitate formed
Table 1. Consequences of the DNA isolate and the criterions in the different chemical trials
For the Dische trial. the reaction depends on the transition of the pentose to the aldehyde which so reacts with diphenylamine to give a blue-colored solution. The strength of the bluish colour is relative o the concentration of DNA. Dische reagent does non organize a blue-colored solution in RNA.
After carry oning the experiment. the group has concluded that insulating Deoxyribonucleic acid from onions is non that easy. Several safeguards like maintaining the temperature to 60 grades must be monitored. and besides the blending of the solution limited to 45 seconds. Several stairss including puting the placing of the solution in an ice bath is besides monitored. likewise with the swirling of the solution. As for the DNA isolate. it reacted largely the same compared to the different criterions used in the chemical trials. It may differ in the strength of the colour from the criterions but still. it yielded positive consequences.
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