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Cario, Shaira A.

Seat # D-8
DNA Isolation
Animal Microbial Sources
(bacteria)
Plant
Preparation of sources

Centrifuge animal blood or sperm
sample at 1000g, room temperature
for 5min.
As for the intact cell preparation to be
embedded in low melting point (LMP)
agarose plugs, wash the cells 2 or
more times by resuspending ice-cold
Phosphate buffered saline solution
with a large-orifice pipette tip,
followed by centrifugation at 1000g,
4C for 5min.
Dispose of the supernatant,
resuspend the cells in 0.5-1.0ml PBS
buffer and adjust to suitable
concentration of cells.
Prepare 10ml of 1% (w/v) LMP
agarose in PBS buffer.
Harvest bacterial cell overnight
culture by centrifugation at 4000g,
4C for 10min and completely
resuspend cells in 50 mM EDTA (ph
8.0) by vortexing.
Resuspend the cells in small volume
(e.g. 1.0 ml) of 50 mM EDTA (pH 8.0)
and adjust to suitable cell of 5 x 10
9

cells/ml.
Prepare 5 ml of 1% (w/v) LMP
agarose in 50 mM EDTA (pH 8.0).
Grind (approx.) 100g of frozen or
fresh tissue into fine powder in
(approx.) 1000ml of liquid nitrogen
with pre-cooled mortar and pestle,
and then immediately transfer the
powder into an ice-cold 1000-ml
beaker containing nuclei isolation
buffer.
Homogenize (approx.) 20g of tissue
each time in 200ml of ice-cold 1x
Homogenization buffer solution in a
blender at speed 4 or puree for 30-
60s.
Filter homogenate into an ice-cold
250-ml centrifuge bottle.
Repeat homogenization and
filtration procedures to complete
remaining tissues, if any.
Add 5-ml of 20% (v/v) Triton X-100
to each bottle containing 200 ml HB
solution; gently mix contents and
incubate on ice for 10min.
Embedding cells in
LMP agarose matrix
Prewarm cells to 45C in a 45C water
bath for (approx.) 5 min.
Prepare a mixture consists of an
equal volume of prewarmed cells and
prewarmed 1% (w/v) LMP agarose in
a suitable buffer using a cut-off
pipette tip.
Aliquot 100l per plug of the cell and
Prewarm cells to 45C in a 45C water
bath for (approx.) 5 min.
Mix the prewarmed cells with an
equal volume of the prewarmed 1%
(w/v) LMP agarose in a suitable buffer
with a cut-off pipette tip.
Using a cut-off pipette tip, aliquot
100l per plug of the cell and LMP
Prewarm cells to 45C in a 45C
water bath for (approx.) 5 min.
Mix the prewarmed cells with an
equal volume of the prewarmed 1%
(w/v) LMP agarose in a suitable
buffer with a cut-off pipette tip.
Using a wide-bore or cut-off pipette
tip, aliquot 100l per plug of the cell
Organism
Steps
LMP agarose mixture into ice-cold
100l plug molds positioned on ice.
Subject the plug molds to incubation
on ice for 10min.
When completely solidified, transfer
agarose plugs into 5-10 volumes of
lysis buffer with 1-2 plugs/ml lysis
buffer.
agarose mixture into ice-cold 100l
plug molds positioned on ice. Allow
the plug molds to incubate on ice for
10min.
When completely solidified, transfer
agarose plugs into 5-10 volumes of
lysis buffer with 1-2 plugs/ml lysis
buffer.
and LMP agarose mixture into ice-
cold 100l plug molds positioned on
ice. Allow the plug molds to incubate
on ice for 10min.
When completely solidified, transfer
agarose plugs into 5-10 volumes of
lysis buffer with 1-2 plugs/ml lysis
buffer.
Extraction of DNA Subject the LMP agarose plugs to
incubation in the lysis buffer for 16-
24h at 50C in a rotary hybridization
oven or in an environmental shaker.
Wash the plugs once in 50mL
Ethylene Diamine Tetra-acetate
(EDTA) for 1h on ice and store them in
50mM EDTA (pH 8.0) at 4C.
Incubate the LMP agarose plugs in the
lysis buffer for 16-24h at 50C in a
rotary hybridization oven in an
environmental shaker with very
gentle vibration.
Wash the plugs once in 50mL EDTA
for 1h on ice and store them in 50mM
EDTA (pH 8.0) at 4C.
Let the LMP agarose plugs be
incubated in the lysis buffer for 16-
24h at 50C in a rotary hybridization
oven in an environmental shaker
with very gentle agitation.
Wash the plugs once in 50mL EDTA
for 1h on ice and store them in
50mM EDTA (pH 8.0) at 4C.
Purification and
quality inspection of
DNA embedded in
LMP agarose
Wash the plugs once in 10-20 volumes
of ice-cold TE and then 3 times in 10-
20 volumes of ice-cold TE containing
0.1mM Phenylmethylsulfonyl
fluoride (PMSF) (1l 100mM PMSF
per 1ml of TE) on ice, each wash for
1h.
Removal of PMSF is important and to
do this, wash the plugs 3 times in 10-
20 volumes of ice-cold TE on ice, each
wash for 1h. The plugs in TE can be
stored at 4C for at least 5 months
without significant DNA degradation.
Evaluate the concentration and
quality of the DNA embedded in the
LMP agarose plugs by digesting them
with 1 or more restriction enzymes
followed by pulsed-field gel
electrophoresis of digested and
undigested DNA against a DNA
molecular weight marker.
Wash the plugs once in 10-20 volumes
of ice-cold TE and the 3 times in 10-20
volumes of ice-cold TE containing
0.1mM PMSF (1l 100mM PMSF per
1ml of TE) on ice, each wash for 1h.
To remove the PMSF, further wash
the plugs 3 times in 10-20 volumes of
ice-cold TE on ice, each wash for 1h.
The plugs in TE can be stored at 4C
for at least 5 months without
significant DNA degradation.
Subject to assessment the
concentration and quality of the DNA
embedded in the LMP agarose plugs
by digesting them with one or more
restriction enzymes; follow this by
pulsed-field gel electrophoresis of
digested and undigested DNA against
a DNA molecular weight marker.
For the washing of the plugs, do this
once in 10-20 volumes of ice-cold TE
and the 3 times in 10-20 volumes of
ice-cold TE containing 0.1mM PMSF
(1l 100mM PMSF per 1ml of TE) on
ice, each wash for 1h.
As removal of PMSF is significant, do
this by further washing the plugs 3
times in 10-20 volumes of ice-cold TE
on ice, each wash lasting for 1h. The
plugs in TE can be stored at 4C for at
least 5 months without significant
DNA degradation.
Assess the concentration and quality
of the DNA embedded in the LMP
agarose plugs by digesting them with
1 or more restriction enzymes; to be
followed by pulsed-field gel
electrophoresis of digested and
undigested DNA against a DNA
molecular weight marker.