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Dna Isolation From Onion

Dna Isolation From Onion

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Published by: Hanz Christian Andrade Mendez on Feb 19, 2012
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, Miranda, M., Maravillias, M., Manlutac, D., Navarro, M.A.,Panaligan, A, Pingol, E., Pique, V.M.,Group 5-2B-Pharmacy
The Experiment was about the isolation of DNA from onion, Ultraviolet measurement of isolated DNAand Chemical characterization of DNA. DNA was isolated form the plant source (onion). The Absorbance of the DNA was then Determined with the use of a Spectrophotometer, the ratio of absorbance was thencomputed. The DNA was then hydrolyzed with hydrochloric acid. The DNA was characterized with the use of Tests for
d Deoxyribose
or The Dische reaction test, Test for Phosphate, Test for Purines a.k.a
Testand Test for Pyrimidine namely as Wheeler-Johnson Test. The test for
produced a brownishsolution while the test for ribose produced a yellowish solution. The test for phosphate gave a colorless withcrystals solution while the test for purines formed a clear solution with yellow precipitate/crystals. Lastly, thetest for pyrimidine gave a purple solution.
Deoxyribonucleic acid is a nucleicacid containing the genetic instructions used in thedevelopment and functioning of all knownliving organisms. DNA is made of chemical buildingblocks called nucleotides. These building blocks aremade of three parts: a phosphate group, a sugar groupand one of four types of nitrogen bases. To form astrand of DNA, nucleotides are linked into chains, withthe phosphate and sugar groups alternating.The four types of nitrogen bases found innucleotides are: adenine (A), , thymine (T), guanine (G)and cytosine (C). The order, or sequence, of these basesdetermines what biological instructions are contained ina strand of DNA.DNA contains the instructions needed for anorganism to develop, survive and reproduce. To carryout these functions, DNA sequences must be convertedinto messages that can be used to produce proteins,which are the complex molecules that do most of thework in our bodies.DNA is found inside a special area of the cellcalled the nucleus. Because the cell is very small, andbecause organisms have many DNA molecules per cell,each DNA molecule must be tightly packaged. Thispackaged form of the DNA is called a chromosome.DNA spends a lot of time in its chromosomeform. But during cell division, DNA unwinds so it can becopied and the copies transferred to new cells. DNAalso unwinds so that its instructions can be used tomake proteins and for other biological processes.Certain tests will be used in order to isolate andclassify the DNA ; Isolation of DNA from onion,Ultraviolet measurement of Isolated DNA, AcidHydrolysis, Dische Test, Test for Phosphates, Test forPurines, and Test for Pyrimidine.
Isolation of the DNA is the first step in theexperiment, The Raw Minced onion is then added withthe Homogenizing solution, which involves breakingdown and removing cell walls and membrane.
When DNA is isolated from organisms,frequently there remains protein present in the DNAsolution; protein is tightly bound to DNA and completeremoval of protein is not possible. To determine theconcentration and purity of the protein it is thereforesubjected for analysis in a spectrophotometer.
DNA is stable to bases, these is one of thefeatures that distinguish DNA from RNA. Strong acids athigh temperatures are capable of breaking down DNAmolecules onto its basic components, at theseconditions both of the phosphate ester bond and the N-glycosides bends between the Deoxyribose, the Purinesand the pyrimidine bases.
Compounds Tested; Samples used and DevicesusedDNA isolation from Onion
The following were used for the isolation of DNA; 2 White/yellow Onions, Homogenizing Solutions;5% SDS( Sodium Dodecyl Sulfate), 0.15 M NaCl, 0.15 MNa
and 0.001 M EDTA, Ice-Cold 95% ethanol.
Ultraviolet Measurement of DNA
The Following were used in the Measurements:Quartz Cuvettes, UV-VIS Spectrophotometer.
Acid Hydrolysis
The Following were used in the Acid Hydrolysis:DNA Sample, 1M HCl
Test for Deoxyribose
The following were used in the test: 3.5mlDiphenylamine reagent, 1.5ml Hydrolyzed DNAsolution, 0.5 ml Standard DNA, Hot water Bath
Test for Phosphate
The Following were used in the Test forphosphates: 1ml conc. H
, 1ml Nucleic Acid Solution,1ml Standard Phosphate solution, 0.5ml conc.HNO
,1ml H
O, 1ml Molybdate Solution, 10ml water.
Test for Purines
The Following were used in the Murexide Test:5-10GTT Nucleic Acid solution, HNO
,10% KOH, water
Test for Pyrimidines
Lastly the following were used in the test: 0.5mlNucleic Acid solution, Bromine Water, BariumHydroxide
Isolation of DNA from Onion.
A 50 ml Homogenizing solution is then preparedin a 125 ml Erlenmeyer flask and was heated in a waterbath until the solution reached 60 deg C, 25 g mincedonion was added to the preheated water bath. A 1.5gpapain was then added and was then kept for 10minutes. After heating place the Erlenmeyer to an icebath for about 5 minutes, after the cooling process,place the solution to in a beaker to be homogenized;after the homogenization process the homogenate wasthen filtered using a cheese cloth into a 250ml beakerand cool it after wards. The cooled solution was thenadded with 15-20ml of ice-cold ethanol, place it on thesides and allow it to drip downwards, a clear layer of ethanol should form on top of the solution, DNA is notsoluble in ice-cold ethanol, when ethanol is added tothe mixture, all the components in the solution exceptfor the DNA stay in the solution, while the DNAprecipitates out. Let the mixture stand for 3-5 minuteswithout disturbing, the DNA can be perceived by awhite precipitate/ strings. Spool the Formed DNA byusing a pre-bent glass pipette, the obtained specimenwas then added with TE buffer solution.
Ultraviolet Measurement of Isolated DNA
The obtained DNA solution (0.5ml) is thenadded to a 4.5 SSC or TE buffer solution. It is thetransferred to a quartz cuvette to determine theabsorbance at the range of 260nm-280nm. Theobtained results are then calculated for the ratio todetermine the concentrations of protein and nucleicacid.
Acid Hydrolysis
Mix the DNA sample with 1M HCl, then heat itup to 100deg C for 60 minutes with occasional agitation,cover the tubes with marbles and placed under arunning water to neutralize the temperature and add
1M KOH. Adjust the PH to 4-6 using Acetic Acid. Use PHpaper to determine the acidity, Centrifuge and add toclean test tubes for chemical Characterization.
Test for Deoxyribose
Add 3.5 ml Diphenylamine reagent to 1.5 mlhydrolyzed DNA solution. The same procedure usedwith the 0.5ml Standard Deoxyribose Solution. Place theprepared mixture in a hot water bath for at least about10 minutes. Cool immediately and observe the results.
Test for Purines
10 drops of nucleic acid solution into a smallevaporating dish, a few drops of conc. HNO3 is thenadded. Carefully evaporate until try in water bath.Moisten the evaporated nucleic acid with some 10%KOH and heat further, note the color changes uponadding the KOH and upon heating. Add a few drops of water then observe the color, then lastly evaporate andnote the color changes.
Test for Pyrimidine
Treat 0.5 ml nucleic acid solution with an excessof bromine water until the solution turns yellow,remove the excess by boiling the solution until it turnsyellow, note the changes, add the excess Ba(OH)
 solution, then test with litmus paper. Take note of theappearance of the solution.
Isolation of DNA and Ultraviolet Measurementof DNA
Isolation of DNA can be done in three stepswhich are Homogenization of deproteinization andprecipitation.In homogenization the onion is minced, thenheated. The heat treatment is done in order to softenthe cell wall and for the breakdown of the cell. Heatingsoftens the phospholipids in the cell membrane anddenatures the ribonuclease enzymes, which if presentswill cause the fragmentation of the DNA and thusprevent it from being spooled.Next is the deproteinization, this processinvolves adding the protease enzyme papain. It is acommon enzyme that removes or prevents proteinsfrom clinging to the DNA. DNA Precipitation is done bymeans of adding ice-cold ethanol. This is done so theDNA will separate from the solution.The homogenizing solution is important in theexperiment, it allows the release of the DNA from theclinging proteins, there are 3 chemicals that are used inthis process, it is Sodium Dodecyl Sulfate, it is adetergent that helps facilitate in the breakdown of thecell membrane. Ethylenediamine tetra acetic acid orEDTA, it also weakens the cell by destabilizing thecations. And lastly Sodium Chloride Facilitates the DNAto precipitate out of the alcohol solution.
Ultraviolet measurement of DNA
Table 1: Isolation and UV measurement of DNA
1.o85(260nm)/0.801(280nm)=1.35 ratio1/0.7=1.43mg/ml24/18=1.33mg/mlThe chart above shows the ultravioletmeasurements of the obtained samples. As shown fromabove, 1.085 at 260nm and 0.801 at 280nm is obtainedfrom the spectrophotometer. At normal condition it hasbeen observed that the normal range for a good qualityDNA sample should have an A
ratio of 1.7-2.0,but we obtained a ratio of 1.35, it can be inferred thatour DNA sample is contaminated or sensitive for it notto be at the normal range. The amount of proteinpresent in the DNA is about 1.43mg/mL, we obtainedthis by dividing 1 with 0.7 we obtained both data fromthe given chart of the amount of proteins present.Lastly the amount of Nucleic acid is 1.33mg/ml, weobtained both of the given data from the given chart.
Chemical CharacterizationsAcid Hydrolysis
Strong acids at high temperatures are capableof breaking down DNA molecules onto its basic

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