Note: Descriptions are shown in the official language in which they were submitted.
This invention relates to a method and an
apparatus for the electrophoresis of a horizontal slab
gel. More particularly, the present invention is
concerned with a simple electrophoresis system for
multiple agarose slab gels. Such a system allows for
example, the separation of multiple DNA samples on four
or more different gel slabs within the confines of a
single apparatus, using only on power supply.
Agarose gel electrophoresis provides a simple,
convenient, and inexpensive way to both analyze and
prepare DNA molecules. Slab gels with multiple slots
are used to perform the numerous assays required in the
development and production of nucleic acid enzymes,
markers, and cloning vectors- Physical mapping and
analysis of DNA genomes as well as studies on the
structure and function of DNA molecules also rely on
such gels D Thus agarose gel electrophoresis has become
an indispensable tool for nucleic acid researchers.
Relatively expensive electrophoresis systems
may be fabricated according to known designs or may be
purchased from different companies. However, the many
useful applications of the technique often generate an
unpleasant shortage of units and/or power supplies in
the laboratory.
~ here are basically two types of electro-
phoresis apparatus: those which operate with vertical
gels and the ones which use horizontal gels. ~lere are
presently available three popular types of apparatus
wherein the gel is in a vertical tube. Bio-Rad
Laboratories has a Model 155 which was designed by
Loening about 1964 and is called a DISC Gel Electro-
phoresis System. With this system, we have an irregular
migration from one tube to the other which means that it
is difficult to compare the results obtained in one tube
with those obtained in another tube, because proper
alignment and adjustment in height should be made which
creates all sorts of difficulties. Another apparatus
sold by Bio-Rad Laboratories is called a Dual Vertical
Slab Gell Electrophoresis Cell. This apparatus is
excellent for use with a polyacrylamide gel. ~owever,
because it is also of the vertical tube type, it is
difficult to adapt it to agarose gels which obviously
do not adhere to glass. In essence, this apparatus is
not practical with agarose gels.
It should be noted that a gel of agarOse is
used when relatively large molecules are involved.
Since polyacrylamide produces a somewhat tight network
it is acceptable for the electrophoresis of small molecules.
However, large molecules do not penetrate into the gel.
When small and large molecules have to be treated simulta-
neously, agarose is preferred, but it should be remembered
that an apparatus which operates horizontally is then
more practical.
The BRL Vertical Gel Electrophoresis System,
model V 161 sold by Bethesda Research Laboratories Inc.
is another apparatus which is available. It should
however be noted that this one also operates as a
vertical tube, with all the disadvantages mentioned above.
Finally, Bio-Rad Laboratories have a horizontal
type of apparatus which is called Model ~O. This device
has to be used with one plate only which means that one
power supply can only treat two gels.
There is therefore a need for an apparatus
which is not complicated but is versatile enough to treat
many plates at the same time by relying on the sarne power
supply.
It is an object of the present invention to
provide a method and an apparatus which overcome at least
most of the disadvantages of the prior art.
It is another object of the present invention
to provide a simple, low cost agarose slab gel electro-
phoresis system which allows the separation of multiple
DNA samples on at least four different slab gels within
the con-fines of a single apparatus, while using only one
power supply.
It is another object of the present invention
to provide an apparatus which enables the electrophoresis
to be carried out with a plurality of horizontal agarose
slab gels.
It is another object of the invention to provide
a hori.zontal slab gel electrophoresis apparatus comprising:
first and second spaced apart vessels, each
adapted to contain a running buffer;
each vessel being provided with an electrode
for connection to a suitable power supply;
at least one horizontal plate to contain a slab
-- 4 --
gel, said plate disposed in bridying fashion over
said first and said second spaced apart vessels,
first means capable of establishing capillary
contact between one end of said slab gel and the running
buffer which is present in said first vessel, and
second means capable of establishing capillary
contact between the other end of said slab gel and the
running buffer which is present in said second vessel.
It is another object of the invention to
provide a method for the separation of components from a
mixture of molecules of mixed molecular weights, wherein a
solution of the molecules is added to wells formed in a
horizontal slab gel and the gel is thereafter treated by
electrophoresis to produce localized bands each being
characteristic of a specific molecular weight. The method
comprises:
- providing first and second spaced apart vessels,
each containing a running buffer and being provided with
an electrode,
arranging at least one slab gel containing plate
so as to horizontally bridge said first and said second
vessels:
establishing capillary contact first between
one end of said slab gel and the running buffer which is
present in said first vessel, and second between the othe~
end of said slab gel and the running buffer which is
present in said second vessel,
connecting both said electrodes to a suitable
power supply: and
visualizing the localized bands produced in said
slab gel.
3~ `
The method according to the invention is
mainly useful for the separation of multiple DNA samples,
in which case the slab gel consists of agarose.
In accordance with a preferred embodiment of the
invention at least one additional slab gel containing plate
is mounted in spaced superimposed parallel fashion above
the one or more slabs already disposed over the two vessels,
and capillary contact is established also between one end
of the one or more additional slab gels and the running
buffer which is present in the first vessel. Capillary
contact is also established between the other end of
the one or more additional slab gels and the running buffer
which is present in the second vessel.
In accordance with another preferred embodiment
of the invention, a plurality of slab gel containing hori-
zontal plates are stac~ed in spaced parallel fashion with
respect to one another and capillary contact is established
between all these plates and the running buffer which is
present in the first and second containers.
In practice, it is preferred although not
essential, that the method according to the invention be
carried out as follows. It is usually applied to the
separation of multiple DNA samples on four different slab
gels and, in this case, it comprises the following steps:
providing first and second spaced apart elongated
containers which can be set in parallel fashion at a
variable distance from one another and each container
having an inside and an outside partition and housing a
removable platinum electrode,
adding a running buffer to each of said
containers, said running buffer to contact said removable
platinum electrode,
preparing at least four agarose slab gels each
having a row of wells formed therein, by pouring an
agarose solution which has been equilibrated at 50C on
at least four horizontal glass plates over each of which
a well-forming comb has been placed parallel to the glass
plate at a distance of about 2 mm from the surface of
said glass plate,
introducing D~A samples into wells of each agarose
slab gel,
arranging at least two agarose slab gel containing
horizontal plates over said first and second containers by
aligning both ends thereof respectively along the inner
partitions of said first and second vessels
covering each end of each of said at least two
agarose slab gels with one edge portion of a sheet of a
two-ply paper wick, and allowing the other edge portion of
each sheet of two-ply paper wick to dip into the respective
running buffer which is present in said first and said second
container'
disposing a pair of horizontal supporting rods
spacedly above said at least two agarose slab gel contain-
ing horizontal plates which are directly arranged over
said first and second containers,
arranging the remaining agarose slab gel containing
horizontal plates over said supporting rods substantially
in alignment with said at least two agarose slab gel con~
taining horizontal plates,
covering each end of each of said remaining agarose
slab gels with one edge portion of a sheet of a six-ply
paper wick, and allowing the other edge portion of each
sheet of six-ply paper wick to dip into the respective
-- 7 --
running buffer which is present in the -first and the
second container'
connecting the removable platinum electrodes
housed in each container to a suitable power supply and
allowing electrophoresis to take place; and
visualizing the localized bands produced in said
agarose slab gel.
In accordance with yet another embodiment of the
invention, a film of electrically insulating plastic
material such as a Handiwrap ~ is inserted between the
ends of the two-ply and six-ply paper wicks which dip into
the running buffer. This makes it possible to regularize
the current which passes through all the agarose slab gels.
In accordance with yet another embodiment of the
invention, before proceeding to the electrophoresis, each
slab gel is first moistened with some running buffer after
which it is covered with plastic film, such as Handiwrap ~ ,
manufactured and sold ~y Dow Chemical~ Covering of the
slab gel with this film enables to prevent dehydration
of the gel during electrophoresis.
In accordance with another preferred embodiment of
the invention, the electrophoresis is carried out for about
4 hours at about 150 ~ and room temperature.
In accordance with yet another embodiment of the
invention, visualization of the localized bands may be
carried out by staining the gels with ethidium bromide in
a buffer. The bands in the gels, such as DNA, may be
visualized by direct illumination with short wave ultra-
violet light. They can be photographed for example with
Polaroid ~ Type 665 film through a Kodak Wratten~ Z3A f,ilter.
Exposure usually lasts 90 seconds.
FIGURE 1 is a perspective view of an electro-
phoresis apparatus according to the
invention, and
FIGURE ~ is a cross-section view of the apparatus
illustrated in FIGURE 1.
FIGURE 3 shows a typical restriction enzyme
concentration assay using four Eco RI
column eluent fractions and ~ D~A as
substrate.
FIGURES 4a, 4b, 4c and 4d show typical restriction
enzyme concentration assay using four
Eco Rl column eluent fractions and ~ DNA
as substrate.
Referring to the drawings, there is illustrated
an apparatus for the electrophoresis of multiple
agarose slab gels which is especially constructed for
allowing the separation of multiple D~A samples on four
different slab gels using the same apparatus and a single
power supply.
The apparatus shown is made mostly of Plexiglas ~.
It comprises two identical elongated box-like containers 3
and 5. Each container is cornpletely open at the top, as
shown in the drawings and has rectangular cross-section
as particularly shown in FIGURE 2. In each case, the
container is made of an inner partition 3a or 5a, and an
outer partition 3b or 5b. As shown, the partitions 3a and
3b and 5a and 5b, are respectively connected together by
means of bottom 3c or 5c, and end portions 3d and 3e, or 5d
and 5e, All the parts just described may all be made of
Plexiglas ~ , although they may be manufactured from other
suitable materials such as Teflon ~
To complete the assembly of each container 3 and 5,
a removable vertical plate 7 or 9 is disposed vertically,
lengthwise with respect to each container 3 and 5, all as
shown in FIGURES 1 and 2. Each vertical plate 7 or 9
3~
defines an electrode support and is provided with one
electrical outlet 11 or 13, each outlet being adapted for
connection to a suitable power supply (not shown) of a
type which is well known to those skilled in the art. ~¢h
vertical plate 7 or 9 has a platinum wire 17 or 19 which
runs from the outlet 13 or 15, along the vertical plate
7 or 9 into the running buffer 21 or 23 which is present
in containers 3 or 5. This arrangement of each vertical
plate 7 or 9 will effectively define an electrode which
is associated with a respective container 3 or 5. m e
vertical plates are in practice made of Plexiglas ~ ,
however Teflon ~ or any other suitable material may also
be used. Plexiglas is a trademark for methyl acrylate plas-
tic while Teflon is a trademark for polytetrafluoroethylene.
In order to make sure that the vertical plates 7
and 9 remain in vertical position, in each container
there are provided a pair of blocks 25 and 27 which are fixed
respectively against the end portions 3d and 3e or 5d and 5e
of each elongated container 3 and 5. This is all well
illustrated in FIGURE 1 of the drawings. Each container 3
and 5 is also provided with a third block 29 which is fixed
against a respective outer partition 3b or 5b. As illustrated
in the drawings, it will be seen that the blocks 25, 27 and 29
are arranged inside a respective container so that the inner
side of a vertical plate defining an electrode support 7 or
9 rests against the pair of blocks 25, 27. On the other hand,
the outer side of an electrode support 7 or 9 rests against
the third block 29. This arrangement enables each electrode
support 7 and 9 to remain in vertical position.
As shown in the drawings, koth containers 3 and 5
are set substantially parallel to one another and are dis-
posed in such a manner on a surface that the distance
-- 10 -
between the two containers 3 and 5 can be adjusted to
vary according to needs.
The device illustrated comprises two agarose
gel slab containing horizontal plates 31 and 33 which,
as shown, are arranged in bridging fashion over the first
and second elongated box-like containers 3 and 5- In
arranging the plates 31, 33 over the containers 3 and 5
care should be taken to make sure that their edges are
parallel respectively with the inner partitions 3a and 5a
of the containers 3 and 5. In the embodiment which is
illustrated7 as it will be seen later~ the outer edges of
the plates 31, 33 extend slightly past the inner partitions
3a, 5a, for a more practical operation of the device accord-
ing to the invention. Although only two plates have been
shown, it is obvious that more plates can be disposed over
the containers 3, 5 depending on the sizes of the plates and
of the containers and the need to carry out a large
number of tests at the same time.
As mentioned previously, the gel 35 which is
formed on the plates 31, 33 has a row of wells 37 which have
been prepared by placing a well-forming comb (not shown)
over each plate 31, 35 at a distance of about 2 mm from the
surface thereof, near the edges of the plates 31, 33 closest
to the container 5.
Once the plates 31, 33 have been placed over the
containers 3,5 a shèet of a two-ply paper wick 39 is arranged
to have one edge portion 41 covering the gel 35 all in
the manner illustrated in the drawings. The other edge
portion 45 is allowed to hang down as shown in the drawings
so as to dip into the running buffer 23 which has been
added to container 5 before initiating the test. Of course
s~
there are used as many ~sheets 39 as there are gels. In
the case which has been illustrated, there are two such
identical sheets 39.
The arrangement which enables to have stacks of
gels will now be described. There are first provided a
pair of elongated bases 49 and 51 which are disposed
transversely at both ends of the apparatus. The upper
face of each base 49, 51 has small vertical holes 53~ 55
and 57, 59 formed therein, the purpose of which will now
be defined. A pair of horizontal supporting rods 61, 63
each being bent at 90 at both ends thereof, are mounted
over the bases 49 and 51 by inserting the 90 ends respect-
ively in holes 53, 57 and 55, 59~ In the arrangement
illustrated, it will be seen that the supporting rods are
spacedly disposed above the two agarose gel slabs 35
which are formed on the horizontal plates 31~ 33, so as
to be parallel with respect to the two gel slabs 35.
The supporting rods 61, 63 hold additional slab
gel containing horizontal plates 65~ Although only one
gel 67 formed on horizontal plate 65 has been shown for
clarity, it is obvious that at least two and often times
more than two gels 67 will be disposed on the supporting
rods if the apparatus is to be used to meet the purpose
for which it was designed~ The gel~)67 is (are) aligned
with the two agarose gel slab containing plates 31, 33
which are arranged in bridging fashion over the containers
3 and 5.
A sheet of a six-ply paper wick 69 is arranged
to have one edge portion 71 covering the gel 67, all
in the manner illustrated in the drawings. The other edge
portion 75 is allowed to hang down as shown in the
drawings so as to dip into the running buffer 47 which
- 12 -
has been added to container 5 before initiating the
test. Of course, once again, there are used as many
sheets 69 as there are gels 67. As mentioned above,
in the case illustrated, only one gel and one paper
wick has been illustrated for the purpose of clarity.
In order to establish electrical contact
between the two electrodes, additional two-ply paper
wicks and additional six-ply paper wicks are provided.
In connection with the gel slabs 35 of the first level,
which are disposed immediately over the two containers
3 and 5, there are provided two sheets of two-ply paper
wick 73 which are arranged exactl~ as sheets 39 but at
the other end of the gel slabs 35, i.e. an edge portion
covers the gel and the other edge portion dips
into the running buffer 21 which has been added to
container 3, before initiating the test.
Turning now to the gel slab(s) which rest(s)
on the supporting rods 61, 63 there is provided an
additional six-ply paper wick which appears at the other
end of the slab 67 and is arxanged exactly as sheet 69,
all as shown in the drawings.
me operation of the device is as indicated
previously with reference to the description of the
method.
- 13
Assay for endonuclease activity
One EcoRI restriction endonuclease unit
is defined as that amount of enzyme required to
completely digest l O~ug of ~ DNA in 1 h at 37 C in a
total reaction mixture of 50 ~1. The eluent fractions
were therefore serially diluted in onefold steps (1:2,
1:3, ..., 1:10), and 5 ~1 of each dilution was added to
separate reaction mixtures containing l~ug of ~ D~A,
100 mM Tris-~ICl, pH 7.2, S mM MgC12, 2 mM 2-mercaptoethanol,
and 50 mM NaCl. The total reaction volume wàs 50 ~1.
After 1 h of incubation at 37C, the reaction was terminated
by the addition of 5 ~1 of a solution containing 5% SDS,
25% sucrose, and 0.05% bromophenol blue. A 20-~1 aliquot
from each of the assay tubes was then loaded onto a 0.8%
agarose slab gel (1~ X 18 X 0.3 cm) prepared in 100 ml
TBE buffer (0.1 M Tris, p~ 8.3, 0.1 M boric acid, 2 mM
~DTA). To prepare the gel, a glass plate is first set up
horizontally on a leveling platform made of a 20 X 20 X 007
cm piece of Plexiglas ~ supported by thxee screws (one at
each front edge, and one at the back), and a well-forming
comb (held at 2 mm above the glass surface by a three-prong
clamp) is placed parallel to the top of the glass plate.
The agarose solution, equilibrated at 50C, is then poured
on the horizontal glassplate in one or two consecutive
steps. After the gel has solidified, the comb is removed
and the samples are applied directly into the wells for
electrophoresis.
- 14 -
3~
Aqarose qel electrophoresis
The electrophoresis unit is constructed from
clear Plexiglas ~ and consists of two vessels (30 X 7 X
4.5 cm) which contain the running buffer and house the
removable platinum electrodes. Electrodes are made from
No. 28 platinum wire and attached to 29 X 6 X 0.2-cm pieces
of Plexiglas ~ (or Teflon ~ ), Before -the run, two
agarose gels are carefully aligned on the inner side of
the facing vessels and are covered at both ends with two-
ply paper wicks made from boiled all-purpose towels
(Johnson & Johnson). The third and fourth gels are
covered at both ends with six-ply paper wicks~ Under
these conditions, migration rate of the DNA molecules is
very similar in the four gels. Plastic film (Handiwrap ~ ,
Dow Chemical) is used to prevent dehydration of the gels
during electrophoresis.
For the present experiments, electrophoresis
was carried out for 4 h at 150 V at room temperature.
After the run, the gels were stained for 30 min with 1
~ug/ml ethidium bromide in buffer. DNA in the gels was
visualized by direct illumination with short wave ultra-
violet light and photographed with Polaroid ~ type 665 film
through a Kodak Wratten ~ 23A filter. Exposure was for
80 s.
RESULTS
Complete digestion of ~ PNA with Eco RI restric-
tion endonuclease generates six DNA frayments which corres-
pond to 44.5, 15.4, 12.1, 11.3, 9.8 and 6.9% of the
total phage yenome. The presence of supplementary bands
with intermediate mobilities indicates partial digestion
of the DNA substrate by ~he enzyme. Based on the
restriction endonuclease unit definition cited above,
the concentration of the undiluted eluent fractions in
- 15 -
enzyme units per microliter may thus be determined by
finding the maximum dilution of the fractions that gives
a complete digest of ~ DNA under standard conditions.
The assay profiles shown indicate that fractions
A - D contain 6, 3, 5 and 3 EcoRI units/~l, respectively.
OnlY five DNA bands are observed in the gels after
complete digestion of the substrate by the enzyme which
results from the comigration of two similar size restriction
fragments. It should be noted that resolution of the DNA
bands in the gels is improved when electrophoresis is
carried out at lower voltage for a longer period of time.
Assay of nucleic acid enzyme activity thus appears as
relatively easy when using the agarose slab gel electro-
phoresis system of the invention~
The system provides equivalent migration rates
in the four large-scale gels to compare relative band
mobilities in numerous samples, and the results are
definitely equivalent to those obtained by other systems
which are more expensive to fabricate or purchase. Because
of its high sample capacity (up to 72 X 25-,ul samples) and
simplicity, less costly equipment (and bench space~ is
required per experiment. The system has been successfully
used for the assay of endonuclease activity in column
eluents and restriction enzyme samples as well as the pre-
paration and analysis of DNA restriction fragments, and
nucleic acid hybridization. It has also proven to be
particularly useful in the screening of sucrose gradient
fractions for DNA nicking-closing enzyme activity~
Another advantage of the system is its
versatility: (1) slabs ranging in size from 2~5 X 7.5 to
28 X 30 cm are currently used in this laboratory, and (2)
simultaneous migration o~ DNA molecules in low- and high-
concentration gels is also possible.
- 16
3~L
~ o particular problem has been encountered while
using this system even though its design requires the use of
paper wicks (instead of agarose wicks) as current carriers.
High buffer holding capacity paper wicks were used to
provide uniform electrical paths between the electrodes and
the gel slabs. Overheating during electrophoresis in
alkaline gels was significantly reduced ~y pumping the
running buffer from one chamber to the other, directly on
the paper wicks.
The electrophoresis system described in this
paper appears as a most effective and useful tool for DNA
separation in agarose gels. Larger units or units designed
for polyacrylamide or polyacrylamide agarose gels are under
construction.
- 17 -
