Quy trình tách chiết DNA genome từ tế bào prokaryote
Quy trình dùng CTAB[sửa]
-
Grow
the
microbe
culture
48hrs
in
a
peptone
broth.
Transfer
20ml
to
a
centrifuge
tube
and
spin.
Decant
the
supernatant.
-
Resuspend
the
pellet
in
600ul
hot
CTAB,
vortex,
and
incubate
1
h
at
65°C.
-
Spin
at
15,000
RPM
for
10
min.
Transfer
the
upper
aqueous
phase
to
a
new
tube
and
add
500ul
of
phenol/chloroform
(1:1).
Mix.
-
Spin
at
15,000
RPM
for
5
min.
Transfer
the
upper
aqueous
phase
to
a
new
tube
and
add
one
more
500ul
of
phenol/chloroform.
Mix
-
Add
1ml
ethanol,
spin
at
15,000
RPM
for
30
min.
at
40C.
Spool
DNA
onto
a
glass
rod
-
Dip
a
rod
into
250ul
absolute
freezing
ethanol
70%.
Dry
naturally
for
5-7h
-
Resuspend
DNA
in
at
least
20ul
TE
0.1X
buffer.
-
Add
20ul
RNase
(1mg/ml)
solution,
and
incubate
1
h.
at
37°C
-
Complete
resuspension
may
take
several
days.
Store
DNA
at
4°C
short
term,
(-)
20
to
(-)
80°C
long
term.
Testing the quality of DNA[sửa]
-
Mix
well
the
DNA
sample
extraction.
Prepare
agarose
0.9%.
Mix
5ul
sample
with
1ul
ethidium
bromate.
Run
with
electrophoresis.
Compare
with
maker.
-
Determine
the
DNA
concentration
at
OD260/280
-
The
receiving
DNA
has
concentration
about
5000ng/ul
and
the
value
of
OD260/280
is
1.8-1.9,
is
good
quality
for
PCR
running.
