EXPERIMENT
8.1
Objective.
To
isolate
DNA
from
available
plant
material
such
as
spinach
leaves,
green
pea
seeds,
papaya
etc
REQUIREMENTS
Plant
material
such
as
spinach
leaves,
green pea seeds
or
green papaya),
mortar
and
pestle,
beakers,
test
tubes,
liquid
detergent, non-iodised
sodium chloride,
distilled water,
meat
tenderizer
or
papain
solution/juice of
papaya/pine
apple
juice, 95
ethanol,
spool etc.
PREPARATION
OF
SOLUTIONS
Detergent
salt
solution
is
prepared
by
adding
10
mL
liquid
detergent
and
10
g
of
non
iodised
sodium
chloride
to 90
mL
of
distilled
water.
Meat
tenderizer
solution
is
prepared
by
adding
5g
of
tenderizer
enzyme)
to
95
ml
of
distilled
water
Juice
of
papaya/pine
apple,
filtered
through
muslin
cloth
can
be
used
as
substitute
for
meat
tenderizer).
5
NaCl
solution
is
prepared
by
dissohving
5
g
of non-iodised
sodium
chloride
in
100
mL
of
distilled
water
Chilling of
ethanol
must
be
done
by
keeping
95
ethanol
in
plastic
bottle
in
the
freerer
over night.
54
cOR
XP RIM NTS
5
PROCEDURE
.Take
5 g
of
the
plant
tissue
(spinach
leaf
green pea
seed/green
papaya)
and
grind
it
in
he
mortar
by
adding
10
mL
detergent,
salt
solution
and
filter
it
through
muslin cloth.
.
Take
10
ml
of
the
filtrate,
add
3-4
ml
tenderizer/papaya
juice
and swirl
the test
tube
by
holding
the tube
between
the
two
hands
to
mix the
contents.
.
Pour
10
mL
chilled
ethanol
carefully
down the
side
of
test
tube
to
forma
layer
on
the
top
of the
content;
let
it
stand
undisturbed
for
about 3
minutes.
Using
the
glass
rod
stir
gently
through
interface
of
the
two
layers
to
collect
the
precipi-
tate of DNA
and
place it in a
test
tube
with
5
NaCl or distilled water.
The
quantity
of
DNA
present
in
the
given
plant
material
can
be
estimated
through
spectrophotometer.
Fig.
8.1.
DNA
that separates
out can
be removed
by
spooling
(spool
=
reel
for
winding
yarn).
OBSERV TION
The
addition
of
ethanol
to
the
solution
causes
DNA
to
precipitation.
The
DNA
fibres appears
as
white
precipitate
of
very
fine
threads
on
the
glass
spoo.
PRECAUTIONS
,
The
plant
material
should
be
washed
throughly
with distilled
water
to
remove any
dust
and dried by
blotting
before weighing.
All
the
glasswares
used
must
be
thoroughly cleaned and
dried.
The
chemicals
and
enzymes
used
for
the
experiment
must
be
of
standard
quality
which
should
be
manufactured by
standard
pharmaceuticals.