Solutions
:
-
Grinding
buffer
:
A
mix
of
extraction
buffer,
nuclei
lysis
buffer
and
5%
sarcosyl
in
a
10:10:4
volume
ratio
.
-
-
Extraction
buffer
(1
liter):
64
g
Sorbitol
12
g
Trizma
base
1
.
7
g
EDTA
Bring
to
pH
7
.
5
using
concentrated
HCl,
keep
at
4
°
C
.
Before
use
add
3
.
8
g
of
sodium
bisulfite
.
-
Nuclei
lysis
buffer
(1
liter):
20
g
CTAB
200
ml
1
M
Tris
pH
7
.
5
200
ml
0
.
25
M
EDTA
Mix
all
of
the
above
.
After
CTAB
dissolves
completely,
add
400
ml
of
5M
NaCl
.
-
Bring
to
pH
7
.
5
using
concentrated
HCl
5%
Sarcosyl
(1
liter):
50
g
N
-
Lauroylsarcosine
-
-
Storage
buffer
(1
liter):
800
ml
95%
Ethanol
100
ml
2
M
Sodium
Acetate
Bring
to
pH
7
.
0
using
concentrated
5
M
NaOH
.
-
TE;
Tris
EDTA
buffer
(100
ml)
1
ml
1
M
Tris
pH
8
.
0
40
m
l
0
.
25
M
EDTA
Bring
to
pH
8
.
0
using
5
M
NaOH
.
Figure
1
demonstrates
the
segregation
of
192
tomato
plants
for
the
PCR
marker
91SP6
.
The
plants
were
sown
in
16
x
8
(columns
x
rows)
trays
and
leaf
disks
from
the
seedlings
were
placed
in
the
96
microtiter
plate
for
DNA
isolation
.
All
plants
were
sampled,
extracted
and
genotyped
on
the
same
day
.
The
genotype
of
the
plants
was
verified
using
a
conventional
RFLP
analysis
with
the
PCR
product
.
The
time
spent
on
extracting
the
DNA
from
384
leaf
samples
(four
plates)
was
only
2
h,
for
one
person
.
This
technique
enables
the
genotyping
of
a
large
population
in
a
short
time
to
identify
rare
genetic
events
.
References
1
.
Deragon,
J
.
M
.
and
Landry,
B
.
S
.
(1992)
PCR
Methods
Appl
.
3,
175
-
180
.
2
.
Geuna,
F
.
,
Hartings,
H
.
and
Scienza,
A
.
(2000)
Anal
.
Biochem
.
278,
228
-
230
.
3
.
Wang,
H
.
,
Qi,
M
.
and
Cutler,
A
.
J
.
(1993)
Nucleic
Acids
Res
.
21,
4153
-
4154
.
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