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GROUP 3

HISTORY
ISOLATION OF DNA FROM ONION BULB
 The very first DNA isolation was done
by a Swiss physician, Friedrich Miescher
in 1869. His method were a little bit
complex hence, simpler methods of
isolating DNA appeared.
 Onions are widely used in DNA
extraction due to its low starch
content which allows the DNA to be
seen clearly.
QUANTIFICATION OF
EXTRACTED DNA FROM ONION
BULB
 Quantification of Nucleic Acids is
commonly performed to determine the
average concentrations of DNA or RNA
present in a mixture, as well as their purity.
 Reactions that use nucleic acids often
require particular amounts and purity for
optimum performance
DIPHENYLAMINE TEST FOR
DEOXYRIBOSE
 Teodoro Gonzalez of the Criminal Identification
laboratory in Mexico City first applied this test in the
United States in 1933.
 The test was performed by covering a suspect's
hand with a layer of paraffin, which, after cooling,
could be broken off and the paraffin treated with
an acid solution of diphenylamine, a reagent used
to detect the presence of nitrates or nitrites.
ISOLATION OF
DNA FROM
ONION BULB
REAGENTS
Homegenizing solution… 5% SDS, NaCl, EDTA
5% SDS a detergent that helps breakdown of the cell
membrane, emulsifies lipids and proteins of cell by
disrupting the non-covalent interaction that hold
membrane together
NaCl allow DNA to precipitate because it shields the
negative charges on the phosphate group of Dna

EDTA weaken cell by complexing to Magnesium and


Calcium needed for membrane stability
ISOLATION OF
DNA FROM
ONION BULB
REAGENTS
Papain protease that denatures protein
95% ice cold ethanol promotes aggregation
of DNA
SSC serve as a buffer solution
DIPHENYLAMINE
TEST FOR
DEOXYRIBOSE
REAGENTS
Diphenylamine reagent diphenylamines that
bounds with the DNA
Conc. Sulfuric acid provide acidic condition
where DNA is converted
QUANTIFICATION OF
THE EXTRACTED DNA
FROM ONION BULB
ISOLATION OF DNA FROM
ONION BULB
PRINCIPLE OF THE TEST
DNA is present in the cells of all living
organisms. This procedure is designed to
extract DNA from onion in sufficient quantity
to be seen and spooled. An onion is used
because it has a low starch content, which
allows the DNA to be seen clearly.
QUANTIFICATION OF DNA EXTRACT

PRINCIPLE OF THE TEST


It allows the quantification of the
extracted DNA extract by
absorbance method using
spectrophotometer.
PRINCIPLE OF THE TEST
 Isolation procedure aims to extract DNA,
from an onion in a sufficient quantity.
 Onion is used because of low starch
content, which allows the DNA to be seen
clearly and be spooled with pipette.
 DNA is (EXTRACTED) 2x
 White precipitate as an indicator,
 White filamentous (MATERIAL ) 2x
 In the upper ethanol layer
 QUANTIFICATION TEST
 It allows the quantification of extracted DNA by
absorbance method using spectrophotometer.
 RESULTS
 The ratio of absorbance at 260nm and at 280nm
is used to asses purity of DNA and RNA. A ratio of
~1.8 generally accepted as “PURE” for DNA; a
ratio of ~2.0, generally accepted as “PURE” for
RNA.
PRINCIPLE OF TEST
 Diphenylamine test for deoxypentose, which is
really present in DNA it is then dehydrated and
converted into reactive w-hydroxylevulinyl
aldehyde.
 *IN ACIDIC CONDITIONS? YES.
 Diphenylamine (MOLECULE)2x
 Will then react to converted sugar
 What to produce? 2x
 The result must have a blue colored complex.
 *In addition to the intensity of color is
proportional to the DNA present.
DIPHENYLAMINE TEST FOR
DEOXYRIBOSE