JPH01138453A - Production of gradient gel for electrophoresis - Google Patents

Abstract

PURPOSE:To improve product quality by supplying a medium liquid for electrophoresis while continuously expanding the liquid in a transverse direction and continuously reducing the same in a thickness direction before the medium liquid flowing from the supply port of a gel forming device arrives at a slot outlet. CONSTITUTION:The gel forming device 8 receives the medium liquid for electrophoresis from a static mixer in the supply port 14 and supplies the liquid from a slit aperture 17 via a slot 16 into a mold consisting of two sheets of glass plates. The supply of the coating liquid from the supply port 14 to the slit 16 needs be continuously expanded and reduced in both the width and thickness at the relatively gentle angles of the expansion and reduction and, therefore, the slot 16 of the forming device 8 has the longer length. The angle alpha of the reduction in the thickness direction is preferably alpha<=0.35 deg.. The medium liquid for electrophoresis is supplied while the liquid is continuously expanded in the transverse direction and is continuously reduced in the thickness direction before the liquid flowing out of the supply port of the gel forming device arrives at the slot outlet, by which the quality of the product is improved.

Description

Detailed Description of the Invention [Industrial Field of Application] Concerning a method for producing a gradient gel membrane for electrophoresis for determining the base sequence of DNA or a partial DNA decomposition product, in particular application of a medium solution for electrophoretic separation. It is about the method.

[Conventional technology]

Conventionally, in flat plate electrophoresis, acrylamide polymer concentration gradient gels, which are not self-supporting, are produced by forming layers of gels with different concentrations between two supports in the direction of electrophoresis, and forming a membrane-like material. It has been used.

However, with the conventional method of forming a layer, it is not possible to produce a gel with a continuous change in concentration. ■ It is necessary to prepare many gel solutions with different concentrations. ■ A high degree of operational skill is required as the gel forming solution must be injected into a narrow mold before it gels. There was a drawback.

In recent years, as an apparatus for manufacturing gradient gels for electrophoresis, a storage tank for a high-concentration gel solution, a storage tank for a low-concentration gel solution, and a mixing section for mixing the two gel solutions, which is comprised of a tank with a variable internal volume, , a gel solution supply path connecting the bottom of the re-storage tank and the mixing section and supplying both gel solutions to the mixing section; a liquid feeding means interposed in the gel solution feeding path; A control means that outputs an operation control signal to control the amount of liquid to be fed for both gel solutions and also outputs an internal volume variable signal to the mixing section before starting the operation of the liquid feeding means; A gel preparation section having a gel preparation chamber at the top, one end opening into the inside from the outside of the mixing section, and the other end opening into the gel preparation chamber from outside the gel preparation section at equal intervals across the entire width of the lower side of the mixing section, and a mixed solution inside the mixing section. Concentration gradient preparation device (1986-
115162) has been disclosed.

[Problem that the invention seeks to solve]

However, when a gradient gel is produced using the above-mentioned production apparatus, (1) the density gradient is not clear because the high-concentration gel solution and the low-concentration gel solution are not sufficiently mixed in the mixing section; (2) Since the amount of the liquid mixture divided by the supply path is not equal, there was a problem that a uniform gel was not formed in the direction of equal molecular weight (width direction) in the gel preparation section.

The purpose of the present invention is to solve the above-mentioned problems, and to provide a stable gradient gel for electrophoresis that is uniform in the molecular weight direction (width direction) and has a continuous concentration gradient with a clear density web in the electrophoresis direction. , to provide a manufacturing method with good reproducibility.

[Means for solving problems]

The object of the present invention is to continuously mix a monomer solution with a concentration gradient and a polymerization reaction initiator solution while supplying the solution to a gel forming device as a medium solution for electrophoretic separation. A method for producing a gradient gel for electrophoresis in which a gel is formed in the gel forming device, the medium liquid being continuously expanded in the width direction and continuously in the thickness direction while reaching the slot outlet from the gel forming device supply port. This is achieved by a method for producing a gradient gel for electrophoresis containing urea as a denaturing agent, which is characterized by comprising a step of reducing and supplying.

In the present invention, a seven-mer solution having a continuous concentration gradient is made from two types of monomer liquids, a concentrated and a light one, whose flow rate ratio is continuously changed. The change in concentration of the monomer liquid is preferably 3 to 35 W/V%.

In the present invention, the mixing method may be carried out using a stirrer in the tank of -C, but mixing and preparation using a static mixer is preferable.

A static mixer is one in which stirring occurs due to the movement of the fluid itself when the fluid is passed through the mixer. Examples of tubular types include spiral tubes, alternating left and right spirals, and propeller shapes with baffles. .

The medium liquid for electrophoretic separation in the present invention may be anything as long as it can produce a medium membrane for electrophoretic separation, and representative examples include raw materials such as acrylamide gel, agarose gel, starch gel, and agar gel. liquid.

Examples of the modifier to be included in the polyacrylamide gel membrane include compounds having at least one carbamoyl group, specific examples of which include urea, formamide, and the like. Among these, urea is particularly preferably used. The amount of the modifier ranges from about 40 wt/v% to 60% based on the volume of the aqueous gel containing the monomer and crosslinking agent.
It is used in the range of wt/v%. When urea is used, the range is from about 6 mol (about 360 g) to a saturated dissolution amount, preferably from about 7 mol (about 420 g) to a saturated dissolution amount for the aqueous gel 11 containing the monomer and crosslinking agent. It can be used in

The thickness of the media membrane is selected depending on the purpose of separation, but is usually 50 mm.
The range is approximately 1.0+m++ from the micrometer, preferably about 0.5am from about 200 micrometers.

In the present invention, providing the liquid medium in the mold of two glass plates by continuously expanding in the width direction from the supply port to the slot outlet and continuously contracting in the thickness direction means that the liquid medium is provided from the supply port to the slot outlet. ■Continuously change the edge line of the medium liquid flow path without abruptly changing the medium liquid flow path.■
When increasing the width, reduce the thickness accordingly ■ This refers to avoiding vortices and stagnation in the overall flow by regulating the angle of expansion and contraction.

Embodiments of the present invention will be described using figures. FIG. 1 is a process diagram of the method for producing a gradient gel for electrophoresis according to the present invention.

The electrophoretic separation medium liquid 12 is prepared by stirring and mixing a low concentration monomer liquid 1, a high concentration monomer liquid 2, and a polymerization reaction initiator 3 in a static mixer 7.

FIG. 2 is an explanatory diagram of the relationship between the flow rate ratios of two kinds of concentrated and light monomer liquids of the present invention. Continuously changing the flow rate means that, for example, initially high concentration monomer liquid 2 is 0L12/l1i.
n, low concentration monomer liquid 1 is 38mj! /win, but if the product length of the gradient gel for electrophoresis is 2, the flow rate is changed as shown in the figure, as shown by the solid line for high concentration monomer solution 2 and the dotted line for low concentration monomer solution 1. , the total flow rate is constant at 38 ml/win, but the concentration changes continuously. Product length 2
At the end of , high concentration monomer liquid 2 is 38 mf/win,
Low concentration monomer liquid 1 is Omj! /win, indicating a highly concentrated film component. In order to change the flow rate ratio in an arbitrary pattern, the flow rate gradient liquid feeding pump 4.5 in FIG. .

On the other hand, the polymerization reaction initiator liquid 3 is controlled by the controller 9b by the constant flow liquid sending pump 6, and a fixed amount is sent to the static mixer 7, where the low concentration monomer liquid 1 and the high concentration monomer liquid 2 are mixed and the polymerization reaction starts. The agent 3 is stirred and mixed to form a medium liquid 12 for electrophoretic separation. The electrodynamic separation medium 12 is supplied to a gel former 8 and then to a mold 13 made of two glass plates.

Next, FIGS. 3(a) and 3(b) are a front view and a side sectional view of an embodiment of the gel forming device 8 of the present invention. The gel forming device 8 receives the electrophoretic separation medium 1 from the static mixer 7.
2 is received at a supply port 14, and is supplied through a slot opening 17 through a thrower 16 into a mold 13 made of two glass plates.

In the case of the present invention, the supply of the coating liquid from the supply port 14 to the slot 16 continuously expands and contracts in both width and thickness;
Since the angle of contraction must be relatively gentle, the length of the slot I6 of the gel former 8 is longer than in the conventional case. FIG. 6 shows a conventional gel forming device.

In addition, in the third enclosure), the angle of reduction in the thickness direction α,
≦0.35" is preferable. FIG. 3(a) is a sectional view of the gel forming device seen from the front, and while the electrophoretic separation medium liquid 12 is being supplied from the supply port 14 to the slot outlet 17, the thrower) 1
6 is continuously expanded in the width direction. Ideally, it is preferable to expand it in a venturi type as shown by the dotted line in the figure, but if you want to simplify the work by considering angular restrictions, it is generally recommended to make the expansion linear with an angle of θ≦40'. It will be done.

Figure 4 shows a mold made of two glass plates.

FIG. 5 shows a state in which two glass plate molds are set in a gel forming device.

[Function] The present invention provides a solution for stirring a static mixer, for example, by supplying a monomer solution having a concentration gradient and a polymerization reaction initiator solution to a gel forming device as a medium solution for electrophoretic separation while mixing the monomer solution and a polymerization reaction initiator solution. Due to the force and self-feeding force, there is no mixing of the liquid before and after the liquid, and the liquid is sent to the gel forming device, making it possible to stably produce a gel with a clear concentration gradient unlike any before.

Furthermore, the present invention continuously expands the medium liquid from the supply port to the slot-like outlet in the width direction and concomitantly continuously contracts in the thickness direction, thereby preventing rapid expansion and contraction of the liquid flow. In order to avoid this problem, specifically: (1) The latitude lines of the liquid flow path are continuously expanded (for example, like a venturi) to avoid eddy currents (stagnation) that occur at the edge lines of the passage. ■ When increasing the width, the thickness is correspondingly reduced to achieve uniform liquid resistance across each channel. ■ Regulate the angle of expansion and contraction, for example, the angle θ≦40° when the liquid spreads;
By setting the angle in the thickness direction to α≦0.35°, generation of vortices is prevented and partial retention of liquid is eliminated.

With these, it is possible to obtain a uniform gel over the entire width.

〔Example〕

Embodiments of the present invention will be described with reference to FIGS. 1 and 2.

Medium for electrophoretic separation Low concentration monomer solution 1 □■ Agarose......65g Urea...4.200g Acrylamide...549g1.3.5-)
Lyacryloyl-hexahydro-3-triazine 5.7g deionized water
Up to 9,0 Ohj! □■ Agarose......40g Urea...4,200g Acrylamide...1,830g1.3.5
-) Liacryloyl-hexahydro-3-triazine...19g Deionized water up to 9. OOOmj! Make a buffer solution □■ Trishydroxymethylamine...121.14g oxalic acid
...65.4g Ethylenediaminetetraacetic acid disodium salt ...7.45g deionized water
Make up to 1,00 (1/!) and add buffer ■...
・・・・・・・・・750m 12.9χ Polyvinylpyrrolidone aqueous solution・80h 125 zN, N, N'
, N'-f) Lamethylethylenediamine liquid...
... 6.7 mj2 mixture - ■ Low concentration monomer ■ 9,000 mj! , high concentration monomer ■
9. OOO+aj! In addition to each of these, a low concentration monomer t&1 and a high concentration upper Zimmer's solution 2 are added.

Next, as the polymerization reaction initiator solution 3, 2χdi-2 ethylhexyl sulfosuccinate solution...100n+ 420.3
75χ Riboflavin phosphate ester sodium salt aqueous solution・150m 13.75χ
Ammonium peroxonisulfate aqueous solution...160m/! Make a mixed solution and put each of the three solutions into a tank.

The sum of the flow rates of low concentration monomer liquid 1 and high concentration monomer liquid 2 is 3811 with flow rate gradient liquid sending pump 4.5! /l1in,
In addition, the flow rate of polymerization reaction initiator liquid 3 is 2.92+/! /wi
n into the static mixer 7 using a constant flow liquid sending pump 6.

In the static mixer 7, the above three liquids are mixed and stirred by a self-blading blade to form a medium liquid 12 for electrophoretic separation.

A medium liquid 12 for electrophoretic separation is supplied from the gel former 8 into a mold 13 made of two glass plates.

The flow rate ratio of the low concentration monomer liquid 1 and the high concentration monomer liquid 2 at this time is as shown in FIG.

The electrophoretic separation medium liquid 12 made in the above manner has a width of 1
BCI was fed into a mold of two 200 μm thick glass plates.

Example-1 At this time, the expansion angle from the supply port in the width direction to the slot in the gel forming device was θ-40°, the thickness reduction angle was α-0,
35', a good quality gel with a uniform concentration in the width direction was obtained.

Comparative Example-1 Gel formation was performed in the same manner as in Example-1 at the same coating solution concentration and temperature using a gel forming device with a spreading angle θ = 45° and a reduction angle α = 0.5°. The concentration distribution became parabolic in the width direction.

[Effects of the Invention] The present invention continuously mixes a 7-mer solution with a concentration gradient and a polymerization reaction initiator solution while supplying it to a gel forming device as a medium solution for electrophoretic separation. A method for producing a gradient gel for electrophoresis in which a gel is formed in a mold, and the gradient gel for electrophoresis can be produced with an accurate, stable, and reproducible concentration gradient by using a static mixer or the like before application. It's now possible. Also, when supplying the medium to the gel forming device, from the supply port to the slot outlet,
By continuously expanding in the width direction and continuously contracting in the thickness direction, the generation of foreign matter due to retention of medium liquid in the gel forming device is eliminated, and it is possible to form a gel with uniform components in the width direction. As a result, product quality has further improved.

[Brief explanation of the drawing]

FIG. 1 is a manufacturing process diagram of the electrophoretic separation medium according to the present invention, FIG. 2 is a concentration gradient of the electrophoretic separation medium according to the present invention, and FIG. 3 is a front view (a) according to the present invention. , side view (b), Figure 4 shows the mold of two glass plates,
FIG. 5 shows a set of two glass plate molds, and FIG. 6 shows a front view of a conventional gel forming device. 1...Low concentration monomer liquid 2...High concentration monomer liquid 3...Polymerization reaction initiator liquid 4.5...Flow rate gradient liquid feeding pump 6...Constant flow liquid feeding pump 7...Static Mixer 8...Gel formers 9a, 9b...Controller 10...Computer linkage adapter 11...
Personal computer 12...Medium liquid for electrophoretic separation 13...Mold 14 of two glass plates...Supply port 16...Slot 17...
Slot opening 1 day... Gel former 19... Supply port 20... Slot 21... Slot exit agent Patent attorney (8107) Seiyuki Sasaki"i

Claims (1)

[Claims] Electrophoresis in which a monomer solution with a concentration gradient and a polymerization reaction initiator solution are continuously mixed and supplied to a gel forming device as a medium solution for electrophoretic separation to form a gel in a mold of two glass plates. A method for producing a gradient gel for use in a gel forming device, comprising the step of supplying the medium liquid by continuously expanding it in the width direction and continuously contracting it in the thickness direction while reaching the slot outlet from the gel forming device supply port. A method for producing a gradient gel for electrophoresis containing urea as a denaturing agent.