A
method
for
DNA
extraction
from
leaves
in
a
96
-
well
format
brew
University
of
Jerusalem,
The
Faculty
of
Agriculture,
P
.
O
.
Box
12,
Rehovot
Eyal
Fridman,
Tzili
Pleban
and
Dani
Zamir
The
He
76100,
Israel
.
FAX:
972
-
8
-
9468265;
Email:
zamir@agri
.
huji
.
ac
.
il
We
olation
describe
here
a
microtiter
-
based
method
for
rapid
DNA
tly
as
a
genomic
template
for
polymeras
is
that
was
developed
for
tom
e
chain
reaction
(PCR)
.
This
method
is
useful
for
CR
amplification
and
for
it
lo
cts
aimed
at
n
(1,
2,
3),
the
96
-
well
format
described
here
can
enhance
day
at
reduced
cost
.
The
method
was
developed
for
mapping
experiments
in
tomato
but
is
o
and
disc
is
ing
extended
t
10
edure
is
channel
pipet,
a
swinging
-
bucket
a
Add
100
m
l
of
grinding
buffer
(using
a
multichannel
pipet)
.
l
roup
successive
plates
before
centrifugation)
.
-
centrifuge
–
4000
rpm
for
15
min
.
-
-
-
to
primer
specifications
.
-
Add
3
m
l
loading
dye
to
the
wells
and
load
15
m
l
of
the
reaction
on
a
1
.
5%
agarose
gel
.
Using
Owl
D3
-
14
horizontal
system
(150
ml
gel
and
four
combs
of
50
wells),
it
is
possible
to
score
the
genotypes
of
2
microtiter
plates
(192
samples)
.
Loading
can
be
performed
using
the
multichannel
pipet
and
it
is
suggested
to
do
so
before
the
gel
is
carefully
submerged
in
the
electrophoresis
buffer
.
ato
from
leaf
tissue
that
can
be
used
direc
large
-
scale
genotyping
performed
in
marker
-
assisted
selection
programs,
the
screening
of
transgenic
plants
by
P
fine
mapping
in
large
segregating
populations
.
This
latter
application
is
necessary
for
high
e
resolution
mapping
of
Mendelian
genes
or
quantitative
tra
ci
(QTL)
in
proj
map
based
cloning
.
Although
several
protocols
are
available
for
small
DNA
extractio
the
productivity
to
hundreds
of
samples
a
also
shown
to
be
efficient
for
other
crops,
such
as
potat
melon
.
A
single
leaf
d
from
each
individual
plant
us
a
single
-
hole
paper
punch
that
is
placed
in
a
96
-
obtaine
flat
-
well
microtiter
plate
.
The
leaf
samples
can
be
kept
in
a
-
80
°
C
freezer
for
periods
and
the
final
DNA
product
is
sufficient
for
at
leas
PCRs
.
The
entire
proc
performed
in
a
96
-
well
microtiter
plate,
using
a
multi
entrifuge
(Eppendorf;
5810)
and
a
96
-
plate
thermocycler
(MJ
PTC
225)
.
c
Protocol
sing
single
-
hole
paper
punch
.
Put
each
disc
at
the
-
Collect
a
single
leaf
disc
per
plant
u
bottom
of
a
well
(flat
-
bottom
microtiter
plate)
.
-
-
Grind
the
leaves
gently
with
a
seed
crusher
(HyPure
HSC
-
200)
using
a
rubber
mallet
(HyPure
SCM
-
100)
.
-
Incubate
at
60
°
C
for
20
min
.
-
While
in
incubation,
take
a
new
96
-
well
microtiter
plate
(with
a
round
bottom)
.
Put
100
m
of
cold
storage
buffer
.
-
Transfer
50
m
l
from
the
leaf
extract
to
the
plate
with
storage
buffer,
mix
by
pipetting
and
put
at
-
20
°
C
for
20
min
(this
stage
can
last
longer
in
order
to
g
Spin
in
a
swinging
-
bucket
Discard
supernatant
and
dry
the
plate
upside
down
on
a
paper
towel
.
Add
50
m
l
of
TE
and
put
in
60
°
C
for
20
min
.
Take
5
m
l
for
a
30
-
m
l
PCR
in
a
96
-
well
microtiter
plate
.
Perform
PCR
according
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