Plaque dot assay

Abstract

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Full text

Volume
14
Number
3
1986
Nucleic
Acids
Research
Plaque
dot
assay
Richard
Powell,
John
Neilan
and
Frank
Gannon
Department
of
Microbiology,
University
College,
Galway,
Ireland
Submitted
31
December
1985
After
primary
screening
of
genomic
libraries
prepared
in
>
,
one
is
sometimes
presented
with
a
large
number
of
potentially positive
plaques.
This
can
arise,
for
example,
when
(a)
a
heterologous
probe
is
used
to
screen
the
bank
under
low
stringency
conditions;
(b)
when
a
long
cDNA
probe
is
used
which
interacts
with
a
large
genomic
tract or
(c)
when mixed
oligonucleotide
probes
are
used.
This
can
give
rise
to
a
complex
secondary
screening
of
these
plaques.
To
circumvent
this,
we
have
developed
a
plaque
dot
assay
described
below
which
is
rapid,
simple
and
compact.
8
Method:
(i)
Mix
0.1
ml
(approximately
10
cell)
of
the
competent
bacteria
with
3
ml
of
molten
soft
(0.7%)
top
agar
and
pour
onto
the
LB
agar
plate.
Allow
it
to
solidify
for
about
10
min
at
room
temperature;
(ii)
resuspend
the
putative
positive
plaque
in
1
ml
of
A
dilution
buffer.
Using
a
1-5
ul
pipette,
spot
on
approximately
0.2
ul
of
phage
suspension.
Allow
this
minimum
volume
to
dry
into
agar.
(iii)
Incubate
at
370C
for
5
hr.
This
results
in
plaque
dots
of
2-3
mm
in
diameter.
Fifty
of
such
dots
can
be
readily
prepared
on
one
plate.
These
are
then
transferred
to
nitro-
cellulose
or
nylon
filters
for
subsequent
analysis
by
appropriate
probes.
Sensitivity:
(1)
Up
to
5
lifts
to
filters
from
a
single
plate
of
plaque
dots
have
been
made
and
successfully
probed.
(2)
A
positive
plaque
diluted
1/20
with
other
plaques
can
be
detected
in
less
than
48
hr
by
standard
autoradiography.
Comments
on
the
method:
(1)
The
0.2
ul
sample
normally
contains
approximately
103-104
pfu.
If
the
titre
is
higher,
the
plaques
become
very
large
with
increased
background.
(2)
If
the
competent
cells
are
allowed
to
grow
to
give
a
lawn
before
adding
the
phages,
the
dots
are
less
well
developed.
(3)
The
method
works
for
M13
as
well
as
A
phages.
(4)
It
is
quicker
and
more
simple
than
DNA
dot
blots.
Uses:
To
date
we
have
used
it
to
(a)
screen
cDNA
probe
selected
plaques
with
different
oligonucleotide
probes,
e.g.
from
the
5'
and
3'
of
a
gene,
to
direct
our
choice
of
plaques
for
detailed
analysis;
(b)
as
a
preliminary
to
secondary
screening
when
the
primary
screening
was
performed
with
probes
for
different
genes;
(c)
to
try
different
hybridization
and
washing
stringencies
on
plaques
selected
by
a
heterologous
probe;
(d)
to
screen
for
different
specific
sequences
subcloned
into
an
M13
vector;
(e)
to
rapidly
reduce
the
number
of
putative
positive
plaques
before
looking
for
the
isolated
cloned
sequence.
Acknowledgements:
The
contribution
of
Emer
Shivnan
to
this
work
is
gratefully
acknowledged.
This
research
was
supported
by
the
Cancer
Research
Advancement
Board
of
the
Irish
Cancer
Society
and
by
the
EEC
Biomolecular
Engineering
Programme.
©)
I
RL
Press
Limited,
Oxford,
England.
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