IV.

Procedure (Schematic Diagram)

A. Extraction of DNA

Homogenize the 500

grams of frozen liver

G1 G2 G3 G4 G5
30 mL 30 mL 30 mL 30 mL 30 mL

Divide 30 mL of liver juice for each group.

 Add 30 mL
of 2 M NaCl
and 30 mL
oF Liquid
Detergent

Heat the
mixture to near
boiling

Filter using cheese cloth

 Collect
the
filtrate

fresh pineapple juice

Pour 40 mL ice cold pure

ethanol gently.

After a second,
white fibers look
like cotton appear
in the ethanol
layer
white fibers
 Spool the
precipitate
and wash
with 10 mL
95% ethanol.

;
;

B.Chemical Characterization of DNA

1. Hydrolysis

10 drops of 60% HClO4

Mix

1/3 of DNA is is isolate

Heat the mixture at 100ºC for

60 minutes with occasional
agitation.
 Centrifuge

1 mL of water

Upon cooling, add

1 mL of water,
grind with a glass
rod to produce a
suspension.

To separate the solution from

the black participate residue.

Keep the clear

supernatant for
chemical
characterization.
 2. Test for Deoxyribose

1.5 mL of the 3.5 mL of

hydrolyzed diphenylamine
DNA solution. reagent

Cool immediately
and observed results.
 3. Test for Phosphate

1 mL of
the nucleic 1 mL of 5
acid M H2SO4
solution.

Heat the mixture over a small

flame, shaking frequently until
the contents of the tube become
brown.

Heat until white

fumes appear.

Add
 Heat
Add 2 more drops of
conc. HNO3

1 mL of water
Colorless
liquid

Heat
 1 mL of 1 M
Ammonium
Molybdate
solution.

4. Test for Purines

Acetate Buffer

1 mL of the 1 mL NaOH 2mL of acetate

solution. buffer
 Add 10 drops of
10% CuSO4

A bluish-
brown
precipitate
will be
formed
Heat

Add 10 drops
of saturated
NaHSO3

Heat in a water bath. A white or

light tan flocculent precipitate will
be observed.
5. Test for Pyrimidines (Cytosine or Uracil)

Treat 1 mL of solution
with 1.5 mL Bromine
water.

Remove excess Bromine by boiling

in a water bath

A purple color 5 M Ba (OH)2 in

indicates Cytosine or excess
Uracil and is due to the
purple Barium salt of
Dialuric acid. The test
is negative for Thmine.