Biochem Oral Report PPT
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Transcript of Biochem Oral Report PPT
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ISOLATION ANDCHARACTERIZATION
DNA
NICOLAS, Jonella JeanPOSADAS, John Arcee
SALCEDO, JenevaUY, Ryan Christopher
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Onion
We use an onion because ofits' cost, abundance and lowstarch content.
Its cell is humongous
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Nucleic Acid
A nucleic acid is a macromolecule composed ofchains of high molecular weight biopolymers ofmonomeric nucleotides. These molecules carry
genetic information or form structures within cells.
The most common nucleic acids are deoxyribonucleicacid (DNA) and ribonucleic acid (RNA). Its functionmainly in the storage and expression of genetic
information
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Deoxyribonucleic Acid
It is the hereditary material in humans and almost allother organisms. Nearly every cells in a personsbody has the same DNA.
Most DNA is located in the cell nucleus (where it iscalled nuclear DNA)
Small amount of DNA can also be found in the
mitochondria where it is called mitochondrial DNA ormtDNA
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Composition of DNA
Sugar is Deoxyribose
Nitrogenous Bases
Phosphate Group
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Nitrogenous Bases
These nitrogenous bases hydrogen bond between opposingDNA strands to form the rungs of the "twisted ladder" or
double helix of DNA or a biological catalyst that is found inthe nucleotides.
Adenine is always paired with thymine, and guanine is alwayspaired with cytosine. Uracil is only present in RNA: replacingthymine and pairing with adenine.
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Purines
Guanine
Adenine
Pyridines
Cytosine
Thymine
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Properties of DNA
Double Stranded
The two strands run on opposite direction
The strands are complimentary and are stabilized byH-bonds between bases (G-C, A-T)
Insoluble in dilute salt solution and ethanol, solublein concentrated salt and water.
attached to a protein (histone) through ionicinteraction (nucleoprotein)
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Nomograph
It is a graphical calculating device, a two-dimensionaldiagram designed to allow the approximategraphical computation of a function.
The result is obtained by laying a straightedge acrossthe known values on the scales and reading theunknown value from where it crosses the scale forthat variable. The virtual or drawn line created by the
straightedge is called an index line or isopleth.
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Objective
To be able to isolate DNA from Onion
To be able to determined the DNA concentration and
PurityTo be able to hydrolyzed DNA by Acid Hydrolysis
To be Able to Characterized DNA that was isolated
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Methodology
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A. Isolation of DNA
from OnionChopped fresh onionsHomogenizingsolution
Ice-cold 95% ethanol
Commercial PapainCheesecloth
Blender
Erlenmeyer Flask
Kitchen Knife
Graduated Cylinder
Funnel
250ml Beaker
DNA spooler
Ice Bath
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50 ml ofhomogenizing
solution in a 250mlerlenmeyer flask
50 mlhomogenizing
solution, 60o
The first few layers ofthe onion was removedThe onion was mincedand weighed to 25g
This was added
50 mlHomogenizingsolution with onion
Solution wasstirred and let sit ina water bath for 5mins, While inwater bath ,
solution wasstirred occasionallyevery 2 mins
50 ml Homogenizingsolution with onion,still in water bath
1.500 g of Crude papain powderwas addedThe solution was kept in thewater bath for an additional 10
mins
Solution washeated to60o
50 ml Homogenizingsolution with onion and1.5 crude papain , still inwater bath
Flask wastransferred intoan ice bath for 5mins
50 ml Homogenizing solutionwith onion and 1.5 crude papain, still in ice bath
Contentswere pouredin a blender
ContentsWereblendedfor 5 mins
Homogenate
A. Isolation and Characterization of DNA
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Homogenate
Homogenate wasfiltered with 4 layers ofcheesecloth into aclean graduatedcylinder
FiltratePrecipitate
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Filtrate
Volume was
measured andtransferred to a250ml beaker
Filtrate in
250 mlbeaker
Beaker was tilted ata 45o angle
95% ethanol(2x homogenatevolume) was poured downthe the inner wall of thebeaker
Solution was leftundisturbed for 2-3minsuntil bubbling stopped
Upper layerethanolLower layer OnionLiquid
With DNA floatingon surface
DNA was collected by placingspooler just below the upperlayer of the liquid and thentwirled it in and out of the 2layers in I direction
Collected DNAWeigh and air dry
The Mixture ofethanol andonion
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B. Determination of DNA
Concentration and PurityStandard Saline Citrate
Tris-EDTA buffer
Spectophotometer
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1.0 mg DNA
Dissolve inSSC solution TE Buffer
Rest of the
DNA precipitateWas setaside forAcidHydrolysis ofDNA
B. Determination of DNA Concentration and Purity
Absorbance was read at 260 nm and 280nmAbsorbance ratio was calculate usingthe equation A260 / A280
A260 = Absorbanceratio of 260nmA280= Absorbanceratio of 280nm
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C. Acid Hydrolysis of
DNA1M sodium hydroxide1M HCl
Medium sized test tube
Boiling water bath
Distilled water
Marble
Filter paper
Graduated cylinder
Funnel
C A id H d l i f DNA
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DNAPrecipitate
DNA was mixed with1.0ml of HCl in a mediumsized test tube
DNA precipitate
mixed with 1.0ml of60% HClO in mediumsi sized test tube
The test tube was coveredwith a marble and
heated at 100o for 60 minsContents of the test tubewas agitated occasionally
Medium sized test tubecontaining solution
Test tube was removedfrom the water bathAllowed to cool in room
temperature
2.5 ml of distilledwater was addedNeutralized with 1M
Sodium Hydroxide
Medium sized Test tube
containing solution + 2.5ml ofdistilled water, Neutral
Particulate matter?NO YES
Store DNA hydrolyzatein refrigerator for DNAcharacterization
Hydrolyzate wasfiltered
Distilled water wasadded to 3ml
3ml of Filteredsoln
C. Acid Hydrolysis of DNA
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D. Chemical
Characterization of DNAD.1 Test for Deoxyribose or Dische ReactionDNA Hydrolyzate
Diphenylamine
Standard Deoxyribose Solution
D.2 Test for Phosphate
DNA Hydrolyzate
Conc. HNO3Conc. H2SO4
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D. Chemical
Characterization of DNAD.3 Test for Purines / Murexide testDNA Hydrolyzate
Conc. HNO3
10% KOHStandard Guanine or Adenine Solution
D.4 Test for Pyrimidines/Wheeler Johnsons Test
DNA Hydrolyzate
Bromine WaterBarium Hydroxide
Standard cytosine or uracil solution
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D. Chemical Characterization of DNA
D1. Test for Deoxyribose and Dische Reaction
0.5 ml of DNAHydrolyzate
0.5ml ofDeoxyribosestandard solution
1.5ml of diphenylamine was added
0.5ml of Deoxyribosestandard solution + 1.5ml of Diphenylamine
0.5 ml of DNAHydrolyzate +1.5 ml ofdiphenylamine
Heated in a boiling waterbath for 10 mins
0.5 ml of DNAHydrolyzate +1.5 ml ofdiphenylamine
0.5 ml of DNAHydrolyzate + 1.5 ml ofdiphenylamine
Results were
observed
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D2. Test for Phosphate
1 ml of DNAHydrolyzate
1ml of Phosphatestandard solution
1ml of conc H2SO4 was added
0.5ml of Phosphatestandard solution +1mlof conc H2SO4
0.5 ml of DNAHydrolyzate +1ml ofconcH2SO4
0.5 ml of conc HNO3 wasadded
0.5 ml of DNAHydrolyzate +1ml ofconcH2SO4 + 0.5ml of concHNO3
0.5 ml of Phosphatestandard solution +1mlof conc H2SO4 +0.5ml ofconc HNO3
l f DNA
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1ml of Phosphatestandard solution1ml Distilled water was added
Heated for 5 mins in a boiling water bath
1ml of AmmoniumMolybdate solution was
added
0.5 ml of DNAHydrolyzate +1ml ofconcH2SO4 + 0.5ml of concHNO3
0.5 ml of Phosphatestandard solution +1mlof concH2SO4 +0.5ml of conc
HNO3
0.5 ml of DNAHydrolyzate +1ml ofconcH2SO4 + 0.5ml of concHNO3 +1ml distilledwater
0.5 ml of Phosphatestandard solution +1ml
of conc H2SO4 + 0.5ml ofconc HNO3 +1ml distilledwater
0.5 ml of DNAHydrolyzate +1ml ofconcH2SO4 + 0.5ml of concHNO3 +1ml distilledwater + 1ml AmmoniumMolybdate solution
0.5 ml of Phosphatestandard solution +1mlof conc H2SO4 + 0.5ml ofconc HNO3 +1ml distilledwater + 1ml Ammonium
Molybdate solution
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1ml of Phosphatestandard solution
0.5 ml of Phosphatestandard solution +1mlof conc H2SO4 + 0.5ml ofconc HNO3 +1ml distilled
water + 1ml AmmoniumMolybdate solution
0.5 ml of DNAHydrolyzate +1ml ofconcH2SO4 + 0.5ml of concHNO3 +1ml distilled
water + 1ml AmmoniumMolybdate solution
Diluted to 10ml with waterSolutiton was let stand for 10 mins
Color of solution andprecipitate formedwas observed
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D3. Test for Purines /Murexide Test
DNA Hyrdolyzatein smallevaporating dish
Standard adenine or guaninesolution in small evaporatingdish
A few drops of conc. Nitric acid, was addedEvaporated to dryness in the fume hood
DNA Hyrdolyzate +Conc. Nitric acid insmall evaporatingdish
Standard adenine or guaninesolution + Conc. Nitric acid insmall evaporating dish
Upon moistening with 10%KOH , theyellow residue became red and thenwas seen with a purplish red hueA few drops of water was addedAnd observations were recorded
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Light yellow Solution Light yellow Solution
Barium Hydroxide was added inexcess.Tested using litmus paper, to verifyIf Basic
Results were then observed
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Results and Discussion
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Isolation of DNA fromOnion
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GRP.Weight ofOnion (g)
Volume offiltrate
(mL)Description of DNA
Weight of air-dried precipitate
(g)
1 25.0377g 40mL Clear yellow solution 0.1241g
2 24.1387g 39mL Clear yellow solution 0.0542g
3 25.07g 45mLTurbid yellowsolution
0.35g
4 25.13g 41mL Clear yellowishsolution
0.1839g
5 26.42g 45mLTurbid light yellowsolution
0.3727
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GRP.Weight ofOnion (g)
Volume offiltrate
(mL)Description of DNA
Weight of air-dried precipitate
(g)
6 25.63g 41mLTurbid of yellowishsolution
0.1938g
7 25.01g 38mLTurbid yellowishsolution 0.24g
8 25.41g 41mL Light yellow solution 0.1102g
9 25.05g 39mL Turbid yellowishsolution
0.0704g
10 23.98g 36mL Light yellow solution 0.2733g
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HeatingSoftens lipid membranes of the cell
Enhances the function of homogenizing solution
Breakdown of cell membrane
Temperature kept at 60C
For maximum activity of the enzyme papain in DNA
deproteinationDNA is degraded at 75C
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Homogenizingsolution
Destroys the cell and nuclear membranes of the onioncells
4 important components:
Sodium Dodecyl Sulfate (SDS)
Sodium citrate
Ethylenediamine tetracetic acid(EDTA)
Sodium chloride
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Sodium Dodecyl Sulfate (SDS)
anionic biological detergent
break down and effectively emulsify the lipid and proteincomponents of the cell
disruption of polar interactions that keep the membrane together
Sodium citrate
chelating agent
causes cellular debris (degraded cell membranes, etc.) toprecipitate for easy filtration
Sodium chloridehelps the DNA to precipitate out of the solution
shields the negative charges of the phosphate group in the DNAbackbone
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Crude Papain Powder
Ethylenediamine tetracetic acid (EDTA)
chelating agentbinds to Mg which is needed for Dnase activity
*Dnase is an enzyme that degrades DNA (undesirable)
For deproteination
Papain enzyme that breaks peptide bonds attach to DNA Optimal temperature range: 60C
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CoolingSlows down DNA breakdown
Swirling even cooling of solution
Blending Further breakdown of cell membranes
45 sec only in order not to destroy DNA molecules
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FiltrationCheesecloth - traps the precipitated cell debris while thesoluble DNA passes through
Ice-cold 95% ethanol Precipitates the DNA
Causes the other components of the filtrate to stay in thesolution
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Determination of DNA
Concentration and Purity
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Group # 260nm 280nm Protein
concentration
Nucleic Acid
Concentration
Absorbance
Ratio
1 0.345 0.372 0.3 mg/mL 8g/mL 0.927
2 0.371 0.373 0.29 mg/mL 7g/mL 0.995
3 0.638 0.523 0.25mg/mL 20g/mL 1.22
4 0.220 0.227 0.22 mg/mL 5g/mL 0.97
5 0.591 0.562 0.4mg/mL 15g/mL 1.05
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Group # 260nm 280nm Protein
concentration
Nucleic Acid
Concentration
Absorbance
Ratio
6 0.183 0.201 0.22 mg/mL g/mL 0.910
7 0.242 0.259 0.30 mg/mL 5.5g/mL 0.9343
8 0.527 0.523 0.35 mg/mL 14.5g/mL 1.0076
9 0.631 0.630 0.48 mg/mL 16g/mL 1.0016
10 1.525 1.335 0.68 mg/mL 50g/mL 1.142
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Light absorbance of DNA
260 nm
Purines and pyrimidines are detected
Light absorbance of proteins
280 nm
Aromatic amino acids are detectedTyrosine, tryptophan and phenylalanine
Good DNA sample:
1.7-2.0 absorbance ratio (A260/A280)Outside this range, DNA solution is contaminated
Lower than expected range: protein contaminated
Higher than expected range: RNA contaminated
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Acid Hydrolysis ofDNA
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Addition of 1M HCl and heating at 100C
To breakdown the double helix structure of DNA
Strong acids at high temperature can break:Phosphodiester bonds
o separates phosphate group from deoxyribose sugar andnitrogenous base complex
-N-glycosidic bonds
o Separates nitrogenous bases from deoxyribose sugar
Hydrogen bonds
o Separates nitrogenous bases pairs
Neutralized with 1M NaOHo acidic condition can affect the results of the characterization
tests
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Chemical
Characterization of DNA
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Standard
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Group # Test forDeoxyribose
Test forPhosphate
Test for Purines Test forPyrimidines
1 Dark purple
solution
Clear Solution
YellowPrecipitate
Yellow Liquid
Orange SolutionRed OrangeResidue
Clear solution
Purple precipitate
2 Dark blue solution Turbid yellowsolutionYellow
precipitate
Yellow precipitateYellow solutionRed Orange
residue
Purple solutionPurple precipitate
3 Dark purplesolution
Clear colorlesssolutionYellowprecipitate
Yellow precipitateYellow solutionRed orangeresidue
Clear colorlesssolution
4 Dark purple
solution
Clear yellow
solutionYellowprecipitate
Yellow precipitate
Yellow solutionRed orangeprecipitate
Purple Solution
Purple precipitate
5 Dark purplesolution
Yellowprecipitate
Yellow precipitateYellow solutionRed precipitate
Purple residue
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Group # Test forDeoxyribose
Test forPhosphate
Test for Purines Test forPyrimidines
6 Dark blue solution Clear yellow
solutionWhiteprecipitate
Red orange solid
formedYellow precipitate
Purple color
solution
7 Dark bluesolution
Light yellowsolutionYellowprecipitate
Clear solutionYellow residueRed residue
Violet residue
PurpleprecipitateColorlesssolution
8 Dark violet solution Yellowprecipitate
White residueYellow solutionBrown yellowsolutionYellow residue
Purple precipitateClear colorlesssolution
9 Dark PurpleSolution
YellowprecipitateColorlesssolution
White solutionYellow residueRed residue
Purple PrecipitateColorless Solution
10 Dark Purple opaquesolution
Clear solutionYellow
precipitate
Clear solutionPurple precipitate
Red OrangePrecipitate
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DNA Hydrolyzate
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Group # Test forDeoxyribose
Test forPhosphate
Test for Purines Test forPyrimidines
1 Light silver solution
White precipitate
Clear solution
WhitePrecipitate
Light yellow
solutionLight brownresidue
Clear solution
White precipitate
2 Clear blue solution Clear light yellowsolutionLight yellowprecipitate
Clear solutionBrown solutionLight yellowishbrown precipitate
Light yellowsolutionWhite precipitate
3 Clear light greensolution
Clear colorlesssolution
Colorless solutionBrown solutionLight yellowsolutionWhite precipitate
Light yellowsolutionWhite precipitate
4 Greenish graysolution
Clear solution
Whiteprecipitate
Brown precipitate
Brown solutionBrown precipitate
Yellowish solution
with precipitate
5 Turbid light bluesolution
Turbid lightyellow solution
Colorless residueBrown solutionBrown residue
Turbid whitesolution
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Group # Test forDeoxyribose
Test forPhosphate
Test for Purines Test forPyrimidines
6 Light blue solution Clear light yellow
solution
White sticky solid
formWhite powder
Colorless solution
7 Light Blue solutionWhite particles
Clear colorlesssolutionNo precipitate
Clear solutionYellow residueYellowish solution
Colorless solutionWhite particles
8 Turbid Solution Clear colorlesssolution
Yellow residueRed solutionPurplish redresidueYellow solution
Clear colorlesssolutionWhite precipitate
9 Clear colorlesssolution
Clear colorless
solution
Colorless solution
Yellow residue
Clear colorless
Solution
10 Light Blue TurbidSolution
No precipitateClear solution
White precipitateClear solution
Light brownprecipitate
T t f D ib
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Test for Deoxyribose orDische Test
POSITIVE RESULT
Blue colored solution
PRINCIPLEDehydration of deoxypentose to
hydroxylaevulinic aldehyde
Complexation of hydroxylaevulinic aldehyde withdiphenylamine (blue solution)
Intensity of blue color is proportional to DNA concentration
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2-deoxyribose
Aldopentose of DNA (RNA- ribose)
Lack oxygen atom at C2
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Test for PhosphatePOSITIVE RESULT
Yellow precipitate
PRINCIPLE
Complexation of phosphate with ammonium molybdate inan acidic medium to form phosphoammonium molybdate(yellow precipitate )
H3PO4 + HNO3 + 12 (NH4)2MoO4(NH4)3PO4.12MoO3
T t f P i
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Test for Purines orMurexide Test
POSITIVE RESULT
Red precipitate
PRINCIPLE
Oxidation of purine by conc. HNO3 forming dialuric acidand alloxan (yellow precipitate)
Condensation leading to formation of alloxanthineAlloxanthine reacts with 10% KOH to form murexide (redprecipitate)
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Test for Pyrimidines
POSITIVE RESULT
Purple color
PRINCIPLE
Bromination of pyrimidines at C5 to producedibromoxhydrouracil (yellow solution)
Alkaline hydrolysis caused by addition of Ba(OH)2 toproduce isodialuric acid
Rearrangement to form dialuric acid and formationof its barium salt (purple precipitate)
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Negative for thymine because it is methylated at C5.
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Conclusion
Many measures have to be taken into account in order to completelyisolate DNA from onion. There are three basic procedure used toisolate DNA: homogenization, deproteination and precipitation.
Purity of isolated DNA can be determined by its protein and nucleic
acid light absorbance with the use of nomograph.Acid hydrolysis was used to hydrolyzed DNA (break the bonds of theDNA) for it to be characterized.
The test for deoxyribose produce a brownish solution, the test forphosphate produce turbid solution with yellow precipitate, the test
for purine produce a red residue, and the test for pyrimidinesproduce purple precipitate which is positive to all the test. However,some of the results from the experiment had a negative resultsbecause of errors. The source of error comes from incorrect use ofreagents.