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Description  |
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FIELD OF THE INVENTION
This invention relates to processes and apparatus for carrying out
horizontal gel electrophoresis for separation and subsequent vacuum
assisted transportation of macromolecules to a support membrane to
facilitate detection.
BACKGROUND OF THE INVENTION
The process known as electrophoresis involves the migration of charged
molecules through a suitable retarding medium under the influence of an
electric field. Generally, the compounds of higher molecular weight
migrate at a slower rate through the medium than do the compounds of lower
molecular weight. Devices have been provided previously for carrying out
electrophoresis. An example of such a device is U.S. Pat. No. 4,415,418 in
which a tray is provided with a raised platform at the center. Removable
partitions are placed in the tray at opposite ends of the platform, and a
conventional electrophoresis gel is poured over the platform to form a
thin layer. When the gel has cooled, the partitions are removed. A comb is
provided to form wells across the surface of the gel. Substances that are
to be subjected to electrophoresis are delivered into each of the wells,
and the tray is at least partially filled with an electrolyte buffer.
Electrodes are positioned at each end of the tray and a sufficient voltage
difference is applied to the electrodes to cause migration of the
molecules of the substance in the wells across the length of the gel,
separated according to their molecular weight. After electrophoresis, the
gel is removed from the original casting tray, and placed in a dish
containing depurination solution. Approximately thirty minutes later this
solution is poured out by tipping the dish toward one edge while the gel
is held with the fingers. It is important to use great care during this
procedure to prevent the gel from breaking because there is no gel support
structure and subsequent processing is possible only with an integral gel.
A denaturation solution is then added to the dish and incubation is
continued for approximately thirty minutes. Again, the solution is
carefully poured off. Then neutralization buffer is added and incubation
is continued for thirty additional minutes.
In accordance with conventional techniques, transfer of the nucleic acids
is accomplished by placing a piece of filter paper, which is as wide as
and longer than the gel, on a platform which is suspended above a solution
of 10.times. saturated saline citrate buffer (SSC). The ends of the filter
paper are long enough to hang off the ends of the platform and dip into
the 10.times. SSC. Thus, the filter paper acts as a wick to absorb the SSC
solution. The gel is removed from the dish and placed on top of the filter
paper saturated with 10.times. SSC. Next, a piece of membrane filter paper
which is the same size as the gel is saturated with 10.times. SSC and
placed on top of the gel. The nucleic acids are eventually bound to the
membrane filter paper. Another piece of saturated filter paper, the same
size as the gel, is placed on top of the membrane. The entire layered unit
is then smoothed to remove any air bubbles that may exist between the gel
and the filter paper. Finally, a stack of paper towels, the same size as
the gel, is positioned on top of the layered unit.
Over a period of about 12 to 16 hours, the 10.times. SSC solution is drawn
up through the gel by capillary action and the nucleic acids are
transferred out of the gel into the membrane above. The paper towels
absorb the excess buffer and provide the force for capillary action. At
the end of the transfer period, the entire layered unit is disassembled
and the membrane is removed for hybridization. This technique is described
in an article by E. Southern, "Detection of Specific Sequences Among DNA
Fragments Separated by Gel Electrophoresis," J. Mol. Biol., 98:503 (1975).
Although the trays such as the one described in U.S. Pat. No. 4,415,418 are
convenient for carrying out electrophoresis, they are not suitable for
situations where a large number of samples must be tested in a relatively
short period of time.
Therefore, the prior art uses a tedious multi-step, multi-apparatus process
for preparing nucleic acid fragments for subsequent hybridization. Four
steps are generally undertaken to achieve preparation of the sample for
hybridization. Electrophoresis was previously described. Depurination
removes purine bases from nucleic acids. Denaturation involves separating
the strands of nucleic acids and breaks down the depurinated nucleic acids
into suitable size to allow eventual transfer of the fragments out of the
gel. Transfer involves allowing the fragments to go out of the gel onto
the porous membrane.
OBJECTS OF THE INVENTION
It is the principal object of the present invention to provide on apparatus
wherein the steps of electrophoresis depurination, denaturation and
transfer to a membrane may all be carried out.
It is an object of the present invention to standardize and simplify the
electrophoresis and transfer techniques to facilitate applications of
molecular biology.
It is a further object of the present invention to provide a process for
electrophoresis and transfer which offers considerable time and cost
savings.
Yet another object of the present invention is to provide an
electrophoretic system wherein a uniform flow rate of buffer solution
above and below the gel surface is maintained, even at low flow rates.
Still another object of the present invention is to provide a more
efficient method for handling the gel and further processing.
Another object of the present invention is to provide an electrophoretic
system including a photoadapter which permits ultraviolet light to be
directed onto a membrane and which permits photographs to be taken of the
membrane.
SUMMARY OF THE INVENTION
In accordance with the present invention, nucleic acid fragments such as
DNA and RNA may be prepared for subsequent hybridization using the process
and apparatus of this invention. The process and apparatus are
particularly useful for detection of gene rearrangements, restriction
fragment polymorphisms and restriction fragment patterns. The process
enables rapid screening of nucleic acids from tissue specimens and body
fluids for the presence of infectious viruses such as Human Papilloma
virus, for typing B-cell and T-cell monoclonal populations, and for
screening patients for the development of cancer or other disease states.
In a first embodiment, the apparatus of the present invention permits
electrophoresis and transfer to be conducted in a single unit. The unit
includes a tray having opposite side walls, opposite end walls and a
bottom wall. There is a central platform in the tray, with a vacuum
chamber between the platform and the bottom wall. The platform surface is
pervious to liquid. A liquid reservoir is provided adjacent each of the
end walls and electrophoresis electrodes are mounted in the reservoirs. A
conduit is provided for transferring liquid into and out of the tray. The
tray may be covered by a lid. The unit is adapted to perform
electrophoresis and vacuum transfer without removing the gel from the
tray.
A second, embodiment of the invention also permits electrophoresis and
transfer to be conducted in a single unit. Like the first embodiment, the
unit of the second embodiment includes a tray having opposite side walls,
opposite end walls, a bottom wall, a central platform in the tray, a
vacuum chamber between the platform and the bottom wall and a platform
surface which is pervious to liquid. An electrode for applying an electric
field is provided in the tray adjacent the end walls. The platform is
located between the electrodes. There is also a conduit for transferring
liquid into and out of the tray. The conduit has a port of a size large
enough to prevent formation of bubbles in the liquid. Unlike the first
embodiment, there is a dam which extends between opposite side walls of
the tray at each end of the platform. The dam has a slot along the bottom
wall of the tray. The tray may be covered by a lid. The unit is adapted to
perform electrophoresis and transfer without removing the gel from the
tray.
The process of the present invention is performed by placing the gel on a
porous platform in a tray. Samples are deposited in spaced wells in the
gel. An electrophoresis buffer is supplied to the tray to cover the gel
and the electrodes. An electric potential is applied between the
electrodes. After the electrophoresis step, depurination and denaturation
are carried out while the gel remains in the tray. In one embodiment, a
transfer membrane is inserted manually between the gel and the platform
before the electrophoresis step. In an alternative embodiment a transfer
membrane is inserted manually between the gel and the platform during the
denaturation step, and the liquid is drawn through the porous membrane to
cause the displaced samples to be transferred from the gel to the
membrane. The membrane is then removed from the tray. Of course, if a
conventional apparatus is used for electrophoresis, the tray of this
invention can be used for transfer of the fragments from the gel to the
membrane by vacuum transfer in accordance with this invention.
DETAILED DESCRIPTION OF THE DRAWINGS
Preferred embodiments of the invention are illustrated in the accompanying
drawings, in which:
FIG. 1 is a top plan view of the electrophoresis transfer tray of the first
embodiment in accordance with this invention, with the lid removed;
FIG. 2 is a side elevational view of the cartridge of this invention,
partially in cross-section;
FIG. 3 is a detail top plan view of the tray with the perforated plate
removed;
FIG. 4 is a cross-sectional view of the tray along the line 4--4 in FIG. 3;
FIG. 5 is a detail view of the plate during the vacuum step showing the
membrane and gel superimposed on the plate;
FIG. 6 is a top plan view of the lid for the cartridge;
FIG. 7 is a cross-sectional view of the lid along the line 7--7 in FIG. 6;
FIG. 8 is a cross-sectional view of the lid along the line 8--8 in FIG. 6;
FIG. 9 is a detail plan view of the tray with the bridge installed;
FIG. 10 is a front elevational view of the tray with the bridge installed
as in FIG. 9;
FIG. 11 is a cross-sectional view of the bridge and tray along the line
11--11 in FIG. 9;
FIG. 12 is a top view of the tray as in FIG. 1 but showing the second
preferred embodiment of the invention;
FIG. 13 is a cross-sectional view of the tray along the line 13--13 in FIG.
12;
FIG. 14 is a perspective detail view of the tray of FIG. 12 showing the
dam;
FIG. 15 is a cross-sectional view of the dam taken along the line 15--15 in
FIG. 12;
FIG. 16 is a cross-sectional view of the tray as in FIG. 15, but showing a
modified form of the dam;
FIG. 17 is a front elevational view of the tray and hood which supports a
camera and ultraviolet light sources;
FIG. 18 is a side elevational view of the tray and hood as in FIG. 17;
FIG. 19 is a cross-sectional view of the connection between the bottom
member of the opaque chamber and the side of the tray taken along the line
19--19 in FIG. 18;
FIG. 20 is a perspective view of a table which can be placed on the support
surface of the tray; and
FIG. 21 is a perspective view of a heat sink plate which can be placed on
the gel surface.
DETAILED DESCRIPTION
Referring to FIGS. 1 and 2, a cartridge 2 is used for carrying out the
electrophoresis and transfer processes of this invention. The cartridge 2
includes a tray 4 and a lid 6. Tray 4 is preferably composed of polyvinyl
chloride or Delrin, a resin which is a registered trademark of E. I.
duPont de Nemours & Co. Inc.; however, any material which has good
dimensional stability for fabrication is non-electrically conductive and
which is chemically compatible with the intended use is suitable. The tray
4 has opposite side walls 8, end walls 10 and a bottom wall 12. A support
surface 14 is provided at the center of the tray. The bottom wall 12
adjacent the end walls 10 slopes downwardly, as shown in FIG. 2 away from
the support surface 14. A plurality of webs 16 extend inwardly from the
end walls 10. The webs facilitate insertion of the gel without tearing.
The webs also align the gel thereby assuring proper orientation of the gel
in the electric field.
As shown in FIGS. 2 and 4, a transverse passage 18 is provided in the
bottom wall 12 and ports 20 communicate with the passage 18 to allow the
circulation of fluid into and out of the tray. A tubing fitting 22 is
provided on the side wall 8. An identical passage 18 is provided at the
opposite end of the tray (FIG. 1) and has corresponding ports 20 and a
tube fitting 22.
Electrodes are provided at each end of the tray. The electrode 24 is in the
form of a thin wire, preferably of platinum, which is wrapped on an
insulated rod 26. The rod is supported in aligned holes in the webs 16.
The electrode 24 extends through the side wall 8 and is connected with an
electrical contact element 28 which is adapted to be connected to a source
of electric potential.
The support surface 14 is formed of a porous plate 30 (FIG. 11). Any porous
plate is suitable, however, a plate made of a porous polyethylene is
preferred. The porous plate should be about 30% to 85% open to
sufficiently pull water. As shown in FIG. 5, the plate 30 has parallel
score lines on each side which are perpendicular, so that small holes
through the plate are formed at the inner section of the score lines. Of
course, any suitable porous plate could be substituted for the plate 30. A
porous membrane 31 has approximately the same dimensions as the plate 30,
so that when it is in place, it substantially covers the plate 30.
Membrane 31 must have properties such that it is optimum for binding of
vacuum assisted transported DNA fragments. Membrane 31 is preferably
comprised of nylon or nitrocellulose such as a nylon membrane comprised of
about 02. to 1.2 micron porosity. However, any fluid-permeable membrane
which is sufficient to bind nucleic acids would be suitable.
A gel is cast on a separate tray as in conventional practice. The gel can
be agarose, polyacrylamide, mixed agarose/polyacrylamide or any other
material suitable for the separation of macromolecules in an electric
field. Generally, 0.7% w/v agarose is used. The gel 32 is then transferred
to the tray 4 where it is superimposed on the plate 30, as shown in FIG.
2. A vacuum chamber is provided beneath the plate 30, as shown in FIGS. 2
and 3. The chamber includes a pair of ridges 34 extending outwardly from a
central channel 36. A tubing fitting 38 extends through the side wall 8
and communicates with the channel 36. The fitting 38 is adapted to be
connected by tubing to a suitable vacuum pump. The opposite ends of the
gel 32, when placed in the tray 4, abut the ends of the webs 16, as shown
in FIG. 11. The side edges of the gel 32 are positioned by engagement with
the side walls 8 of the tray.
As shown in FIG. 6, the lid 6 has a shoulder 40 which extends around the
perimeter of the lid and engages the inside surface of the side walls 8
and the end walls 10. A sealing gasket 42 is retained within a groove in
the shoulder 40. The gasket 42 prevents the leakage of fluid from the
interior of the cartridge. A gas bubble channel 44 is formed in the
interior of the lid 6 and extends along the end walls and side walls of
the tray. The channel portion at the right side of FIG. 6 is more shallow
than the groove at the left side of FIG. 6, and the grooves extending
along the side walls 8 progressively increase in depth from the right end
to the left end as viewed in FIG. 6. This arrangement of the grooves
causes the gas bubbles to migrate progressively toward an outlet port 48
through the lid 6. The outlet port has a tube fitting 48 through which the
gas bubbles can escape.
The central portion of the lid 6 has a plurality of grooves 50 formed in
the top side of the lid. As shown in FIGS. 6 and 8, ports 52 extend from
the bottom of the grooves to the lower surface of the lid so that fluid
can pass from the grooves 50 into the interior of the cartridge when the
lid is in place. A cover plate 54 encloses the grooves 50 and a tube
fitting 56 allows liquid to be conducted through the plate 54 into the
interior of the grooves 50.
In order to enable samples to be delivered into the wells formed in the gel
layer 32, a bridge 58 is provided. The bridge aids in directing the
operator to the correct well for sample filling thus making it easier to
fill the well. The bridge is received in vertical slots 60 in each of the
side walls 80 at a position that is aligned with the wells that are molded
in the gel layer. A black strip may be positioned near the wells to aid in
visualizing the wells during sample loading. The bridge 58 includes
funnel-shaped passages 62 in a shape to receive the tip of a pipette for
delivering the samples into the wells formed in the gel. As shown in FIG.
10, arches 64 are formed in the bridge between the passage 62 to allow
fluid circulation between opposite sides of the bridge. If additional
recirculation is necessary, the bridge can be removed prior to
electrophoresis.
In operation, a gel layer is formed in accordance with conventional
practices of a proper size and shape to fit within the tray 4, so that the
ends abut the ends of the webs 16 and the sides of the gel fit between the
side walls 8 of the tray. Preferably, the gel is cast in a separate tray
and has a series of wells molded in the gel layer adjacent on end. The gel
32 is then removed from the casting tray and placed on the porous plate
30, so that the gel 32 occupies the position shown in FIGS. 2, 9 and 11.
The sloping top edge of each web 16 aids in guiding the gel into position
in the tray 4. A source of electric potential is connected with the
electrical connectors 28 and a system for circulating fluid is connected
with the tubing fittings 22. An electrophoresis buffer is added to the
tray to a depth that fully covers the electrodes 24 and the gel. The
bridge 58 is installed in the slots 60 after the gel layer is positioned
on the support surface 14. The samples are then delivered into the
passages 62 from which they pass into the individual wells. The bridge
remains in place when the lid 6 is applied. The electrophoresis buffer is
recirculated through the passages 18 by the use of a conventional pump, to
provide a fluid current passing over the gel. At the end of
electrophoresis, the electrophoresis buffer is then withdrawn from the
tray and a depurination buffer is pumped into the tray. After a
predetermined period of time, the depurination buffer is then pumped out
of the tray and a denaturation buffer is pumped into the tray. The gel
tends to float in the denaturation buffer, and before the vacuum is
applied, the membrane 31 is inserted manually into the space between the
gel and the plate 30, as shown in FIGS. 5 and 11. The gel is then lightly
pressed against the membrane and held in place by appropriate means, such
as a band or tube 39, as shown in FIGS. 9 and 11. After a suitable elapse
of time, the denaturization buffer is withdrawn through the vacuum fitting
38 at a relatively slow rate, which transfers the displaced samples from
the gel onto the permeable membrane 31. As an alternative, water, or
denaturation liquid may be sprayed onto the surface of the gel through the
tube fitting 56 as the liquid is being withdrawn through the tube fitting
38. The lid 6 is then removed and the permeable membrane 31 may be removed
from the tray for further processing. The membrane now contains the
displaced samples in preparation for hybridization.
The cartridge of this invention has the important advantage that it allows
electrophoresis and transfer to occur without having to remove the gel
from the tray. At the completion of the operation, the nylon membrane
bearing the samples can readily be removed for subsequent treatment.
The electrophoresis apparatus is designed to accommodate either a large
number of analytical samples or milligram quantities of fragments for
preparative runs. Typically, the number of samples which may be introduced
into the cartridge may range from about 5 to 15. Generally, 10 samples
plus two controls have been found to be suitable.
The tray 4 and the lid 6 must be compatible with standard electrophoresis
and nucleic acid transfer reagents. Typical reagents include up to 3 molar
(M) salts, acetic acid, 1 M hydrochloric acid and 0.5 M sodium hydroxide.
Many polymers could be suitable for the present invention. Furthermore, in
view of the direct current being used during electrophoresis, the tray 4
and lid 6 should not conduct electricity. The lid 6 is preferably
plexiglass (acrylic) since a further advantage is obtained with the use of
a clear cover since it would allow visual tracking of optional dyes during
electrophoresis.
Using one buffer or solution throughout the foregoing procedure is more
efficient and economical than using a different buffer or solution during
each of the electrophoresis, depurination, denaturation and transfer
stages. Alternatively, four separate solutions may be used in the practice
of the present invention. For example, during electrophoresis any buffer
well-known in the art is suitable such as any solution of a weak acid or
base and its salts, such as acetates, borates, phosphates and phthalates,
which behave as buffers. Typical compounds used in preparing buffers
include acetic acid, phenylacetic acid, sodium acetate, ethylene diamine
tetraacetic acid (EDTA), phosphoric acid, boric acid, hydrochloric acid,
sodium hydroxide, sodium chloride and the like. During electrophoresis, a
buffer comprised of 40 mM tris-acetate, pH 8, 12 mM sodium acetate and 2
mM EDTA, pH 8, is preferred.
During depurination, any solution which chemically assists in depurination
or depyrimidination would be useful. These solutions are well-known in the
art. A buffer comprised of 0.25 M hydrochloric acid can be used. It was
discovered that during depurination, it is important to expose the lower
surface of the gel as well as the top and side surfaces to the solution.
For this reason, the membrane 31 is not inserted between the gel and the
platform until just before applying the vacuum under the porous plate 30
to transfer the cells to the membrane.
During denaturation, any solution which assists in breaking the hydrogen
bonds between the nucleic acid strands is suitable. These solutions are
also well-known in the art. For example water and heat may provide
satisfactory results, also formamide or any alkali such as sodium
hydroxide or potassium hydroxide. In the present invention a solution of
0.5 M sodium hydroxide is preferred.
As the transfer solution, any solution which allows transfer and binding of
the nucleic acid strands to the membrane would be suitable. These
solutions are also well-known in the art. Advantageously, in the practice
of the present invention the transfer solution is preferably 0.5 M sodium
hydroxide, the same as the denaturation solution.
The time periods used in each of the above-described stages may vary over a
wide range depending on the processing conditions. For instance, each of
electrophoresis, denaturation and depurination may require from about 10
minutes to 5 hours. Useful techniques to decrease the amount of time
required for processing include increasing the voltage, using larger
ports, selecting the optimum thickness of the gel, different sizes of
membranes and the support plate, and the like. A particular advantage of
the present process is that the time required for electrophoresis is
approximately 40-65% of the typical time required for electrophoresis
using prior art apparatus and techniques. A significant time savings is
offered by the present invention because of the circulation of fluids,
i.e., buffers, through the electrophoresis transfer cartridge, thereby
allowing a constant pH and temperature to be maintained during the
process. In addition, the geometry of the cartridge results in
concentrating the electric field within the gel which also speeds the
process.
Furthermore, by maintaining a relatively frequent circulation of the
buffer, a smaller volume of buffer is suitable in contrast to the buffer
requirements of prior art electrophoresis processes.
The transfer step may require from about 10 minutes to two hours.
Generally, about 60 minutes produces adequate results. This step also
offers a considerable time savings over prior art transfer techniques.
Capillary transfer, for instance, requires about 12 hours, squash blot
transfer requires about 3 hours and standard electrotransfer requires
about four hours.
A further advantage of the present invention is the time and labor savings
that results since the apparatus is a cartridge. The decrease in the
number of mechanical steps to be performed by a technician o operator
assists in maintaining the accuracy of the procedure since a decrease in
the number of necessary steps to be performed also minimizes error.
Other components are useful to achieve the objects of the present invention
in addition to the above-described electrophoresis transfer cartridge. For
instance, a microprocessor controller may be used to automate
electrophoresis and DNA/RNA transfer. Such a controller would control the
voltage and time for electrophoresis, the valves for reagents, the pumps
to add and remove reagents and the vacuum system for DNA transfer. The
electrophoresis transfer cartridge may be connected to a variety of
standard laboratory equipment including peristaltic pumps.
The following example is intended to demonstrate one method that may be
used to practice the present invention. The following is not intended to
limit the invention in any way.
EXAMPLE
Electrophoresis Transfer Process
A 14 cm long.times.11 cm wide.times.0.65 cm deep 0.7% w/v agarose gel was
placed and aligned in a polyvinyl chloride electrophoresis transfer tray
having inside dimensions of 20 cm long.times.11 cm wide.times.2 cm deep.
The gel rests on a liquid pervious platform or support plate made of
porous polyethylene. Approximately 200 ml of an electrophoresis buffer
comprised of 40 mM tris-acetate, 12 mM sodium acetate and 2 mM EDTA was
added by hand.
Restriction enzyme digested human genomic DNA samples were loaded into the
wells and a plexiglass top was placed over the polyvinyl chloride tray.
The unit was then plugged into a power supply which was set at 90 volts.
The power was turned on. Electrophoresis was allowed to continue for
approximately ten minutes. Then the buffer was recirculated at a rate of
approximately 25 ml/min for the duration of electrophoresis.
Electrophoresis continued for approximately two hours. The power was
turned off and the electrophoresis buffer was pumped out.
Approximately 200 ml of a depurination solution comprising 0.25 M
hydrochloric acid was pumped into the polyvinyl chloride tray. The
solution was allowed to stand for approximately 15 minutes and then the
solution was pumped out. Next, approximately 200 ml of a denaturation
solution was added to the polyvinyl chloride tray. A nylon membrane was
inserted between the gel and the porous plate. A vacuum pump was turned on
to withdraw fluid through the porous polyethylene plate at about 2 ml/min.
This was continued for 60 minutes to allow the DNA to transfer from the
gel to the nylon membrane below the gel. The unit was turned off.
A second preferred embodiment of the invention is illustrated in FIGS.
12-21. Instead of the plurality of ports 20 (FIG. 1) communicating with
the passage 18 to allow the circulation of fluid into and out of the tray,
the modified tray 4' as shown in FIG. 12, has a single port 20' at each
end of the tray. Each port 20' has a diameter greater than that of each of
the individual ports 20 of the first embodiment as shown in FIG. 1. A dam
17 is mounted at each end of the tray 4'. The dams 17 extend between the
opposing side walls 8'. A plurality of webs 16' on the bottom wall 12'
provide further support for the dam. Both of the dams have a transverse
slot 21 (FIGS. 13 and 15) which extends substantially the full length of
the dam.
The use of a single relatively large port 20' in combination with a dam 17
and slot 21 ensures uniform flow of liquid across the gel in
electrophoretic systems where the liquid flow rate is typically small. The
webs 16' also assist in avoiding lateral currents while the liquid is
flowing. In the systems of FIG. 1, the liquid must be supplied at a
relatively high pressure through the fitting 22 in order to obtain a
uniform flow of liquid through each of the ports 20. At the low pressures
associated with electrophoresis, therefore, there can result a higher flow
rate of liquid across one portion of the gel than across another portion
of the gel. In severe cases, this could affect the migration of the
samples during electrophoresis, lead to overheating of the gel and
disruption of the pH levels at those areas of the gel where the flow of
buffer is insufficient.
The presence of a single larger port and a dam provides a uniform flow of
material even with low pressure flows. The dam 17 forms a chamber 23 at
both ends of the tray 4'. In these chambers, the turbulence or velocity
pressures generated by the inflow and outflow of fluid is dissipated, and
only a very minute head differential between the opposite sides of the dam
17 causes flow through the horizontal slot. The slot 21 can be either at
the bottom of the chamber 23 as shown in FIG. 12, or part way up, but it
must be entirely covered by the liquid surface. Turbulence in the end
chambers is minimized by having the input and output supply systems as
large as possible and by directing the input flow upward where the energy
can be dissipated as waves on the surface. If the process fluid is
suitable, open cell foam structures can be inserted in the chambers 23 to
further dampen turbulence.
Another advantage of using a single larger port and a dam as opposed to a
plurality of small ports to distribute a buffer liquid relates to the
formation of air bubbles. More specifically, as buffer passes through the
ports 20 into the tray, air bubbles can be carried along with the buffer.
These air bubbles can collect until enough pressure builds up within the
system so as to disrupt the flow of additional buffers into the tray. This
problem may be so severe that it is necessary to incorporate specialized
control systems within electrophoretic circulating equipment to effect
removal of the entrained air and resumption of the buffer circulation. Due
to changes in the physical chemistry of a fluid flowing through a single
large port as opposed to a plurality of smaller ports, the present system
eliminates bubble formation within the tray and thus concomitantly
eliminates the need for special control equipment. The use of a larger
buffer fluid port is also advantageous in that it enables particles
carried by the circulating fluid to pass through easily.
If it is desired to have other than uniform flow across the width of the
gel, the slot can be modified to accomplish the desired objective and the
adjustments are not unduly sensitive. For example, if it were desired to
have a greater flow along the two outer edges in order to accomplish
additional cooling, the slots could be widened at the edges as shown in
FIG. 16.
The ports should be of size large enough to avoid bubble formation in the
circulating fluid. It has been found that a hole size of 3 to 5 mm is
satisfactory for this purpose. Although the port is shown near the side
wall 8' of the device, it may be placed at any mechanically feasible
location behind the dam 17. The horizontal slot formed by the dam and the
bottom of the tray should be of a size large enough to permit passage of
insoluble materials and small enough to generate uniform flow. For
example, the slot may be between 1 and 5 mm in height.
As illustrated in FIGS. 14 and 15, three electrodes 24' are advantageously
mounted on the surface of the dam 1 | | |
