JPS63298045A - Production of gradient gel film for electrophoresis - Google Patents

Abstract

PURPOSE:To obtain a titled film having a uniform performance in a transverse direction as well by feeding monomer liquids of a high concn. and low concn. at continuously varied flow rate ratios,adding and mixing the polymn. reaction initiator liquids corresponding thereto, supplying and mixing the mixtures to the respective supplying ports of a coating head and coating this liquid mixture from a slot-shaped aperture onto a web. CONSTITUTION:The low-concn. polymer soln. 12a and the high-concn. polymer soln. 12b are supplied from the respective supplying ports in the coating head 8 and the liquids are expanded in the transverse direction of coating by cavities 13a, 13b. These liquids are press-fed by respective distributing capillaries 14a, 14b to a mixer 16. The directions of the liquids are defined by a partition plate 15 between the capillaries 14a, 14b at this time The two solns. 12a, 12b are mixed by the mixer 16 and the soln. mixture is entered as a medium liquid for electrophoretic sepn. into the slot 17. The liquid mixture is coated from the slot aperture 18 onto the web 19 on a coating roller 20. Since the liquid resistance of the capillaries 14 with the mixer 16 is large, the distribution in the transverse direction of the coating liquid in the cavities 13a, 13b is uniformly executed and foreign matter, etc., are completely dissolved, by which the distribution to a uniform film thickness is executed.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a method for producing a gradient gel membrane for electrophoresis for determining the base sequence of DNA or a partially degraded DNA product, particularly for electrophoretic separation. The present invention relates to a coating head that applies a medium liquid to a web.

[Conventional technology]

Conventionally, in plate electrophoresis, acrylamide polymer concentration gradient gels that are not self-supporting are layered in the electrophoresis direction using gels with different concentrations on one support or between two supports. It has been used as a film-like material.

However, the method of forming and using a gel on a support is
When forming a gel on a support, when setting it in an electrophoresis tank,
While storing the gel or adding an analytical sample, it is possible to accidentally break the gel or drop something other than the sample onto the gel, damaging the gel. was necessary.

On the other hand, in the vertical electrophoresis method, in which a mold is made from two glass plates, etc., a gel is formed in the mold, and the electrophoretic analysis is performed while the mold is held vertically, the thickness of the mold is made uniform. It was difficult to do this, and required a high degree of skill in freezing operations, as the gel forming solution had to be injected into a narrow mold before it gelled.

Particularly in DNA base sequencing operations, it is desirable to create long gels so that as many DNA fragments as possible can be analyzed in one gel, but such gels are difficult to manufacture and handle. Furthermore, since a glass plate is used, there is a drawback that the glass plate is easily damaged.

In recent years, as a method for industrially producing gradient gel materials for electrophoresis, an aqueous solution or aqueous dispersion of a mixture of an acrylamide monomer and a crosslinking agent is prepared, and a free radical generating substance that absorbs light irradiation and initiates polymerization of the monomer is used. The solution is then formed into the desired gel product, and the formed solution is irradiated with light to polymerize and crosslink the monomer solution or dispersion. A method for producing an electrophoretic gradient gel, which includes a step of adjusting the light irradiation time applied to the solution. (Unexamined Japanese Patent Publication No. 61-28
Furthermore, two types of aqueous solutions containing a monomer, a crosslinking agent, and a polymerization initiator and having different concentrations are introduced into a molding machine while being mixed while gradually changing the mixing ratio. A method for producing a gradient gel for electrophoresis, which completes polymerization of a monomer and a crosslinking agent in a container and has a polymer concentration gradient in the direction of electrophoresis. (Special Publication No. 61-22903, Special Publication No. 61-22903, Publication No. 61-22903,
39617) etc. have been disclosed.

Further, a general coating head as shown in FIG. 6 has been used as a coating head for coating the electrophoretic separation medium onto a continuously running web. That is, the electrophoretic separation medium liquid as the coating liquid 22 is pumped by the metering pump 24 to the cavity 23 in the throat 29. The cavity 23 is a slot that extends to the full coating width and communicates with the slot opening 28. This is a liquid reservoir that supplies the coating liquid to the slot 27. The coating liquid flows from the cavity 23 into the slot 27.
The coating material enters the web 19 on the coating roller 20 through the slot opening 28 . At this time, a differential pressure chamber 2 is used to create back pressure behind the coating bead to stabilize coating.
1 is used.

[Problem that the invention seeks to solve]

However, the former first manufacturing method suffers from high costs due to poor light irradiation equipment and poor productivity, and poor resolution due to the thick gel. There are problems such as a cross-linking reaction occurs, making it impossible to obtain a stable and reproducible gradient gel. When the liquid is branched into the molding machine, it is difficult to branch the liquid uniformly, and a reproducible gradient gel cannot be obtained.

Furthermore, with regard to the coating method, the uniformity of the film thickness in the width direction of the coated product was not sufficient, and furthermore, foreign matter was generated due to long-term operation, so it was necessary to disassemble and clean the coating head.

The purpose of the present invention is to solve the above-mentioned conventional problems, and to reproduce stability with a uniform coating thickness over the entire coating width, no generation of foreign matter, and a continuous concentration gradient in the running direction of the web. An object of the present invention is to provide a method for producing a gradient gel membrane for electrophoresis which has good properties and productivity.

[Means for solving problems]

The object of the present invention is to provide a method for producing a gradient gel membrane for electrophoresis in which a medium solution for electrophoretic separation is continuously applied onto a continuously running web, and a method for producing a weak solution of the medium solution for electrophoretic separation. First, the high-concentration monomer solution and the low-concentration monomer solution are fed by continuously changing the flow rate ratio, and the corresponding polymerization reaction initiator solution is added to and mixed with each solution, and the solution is applied to each of the coating heads. Electrophoresis characterized by supplying the two liquids to a supply port, then mixing the two liquids in a coating head using a mixing device, and applying the mixed liquid as a medium liquid for electrophoretic separation onto a web from one slot-shaped opening. This is achieved by a method for producing a gradient gel membrane for

The web in the present invention may be made of any material as long as it is in the form of a sheet with good flatness, is non-conductive, and is substantially water-impermeable (e.g., polyethylene terephthalate, polycarbonate of bisphenol A, Vinyl polymers such as polyester, polymethyl methacrylate, polyethylene, polystyrene, polyvinyl chloride, polyamides such as nylon, and their copolymers (e.g.
Vinylidene chloride/vinyl chloride copolymer) is preferably used.

The electrophoretic separation medium liquid in the present invention is a solution that is a raw material for an electrophoretic separation medium membrane, and representative examples include acrylamide gel, agarose gel, starch gel, agar gel, cellulose acetate porous membrane, A liquid that is used as a raw material for filter paper, etc.

The thickness of the media membrane is selected depending on the purpose of separation and is usually 50 μm.
Approximately 1. Qw, preferably 200 for gel media membranes
μm to approximately 9.5 mm, approximately 7 in the case of porous membranes and filter paper
The range is from 0 μm to IH.

Continuous coating methods in the present invention include slide bead coating and extrusion coating.

Hopper coat, curtain coat, etc. are used.

Embodiments of the present invention will be described in detail using figures.

FIG. 1 is a process diagram of one embodiment of the method for producing a gradient gel membrane for electrophoresis according to the present invention.

In FIG. 1, a low concentration monomer solution 1, a high concentration monomer solution 2, and polymerization reaction initiator solutions 3a and 3b are dissolved and prepared in respective tanks. l! , 1 and the high-concentration monomer liquid 2 are supplied to the flow rate gradient liquid feeding pumps 4.5., 1 and 2, respectively. is pumped to each stirring device. In this case, static mixers 7a and 7b are most suitable as stirring devices. At this time, low concentration monomer solution 1 and high concentration monomer solution 2
is controlled by the controller 9a via the computer linkage adapter 10 in accordance with instructions from the personal computer 11 so as to continuously change the flow rate ratio.

An example of continuously changing this flow rate ratio is shown in FIG.
The high concentration monomer solution 12b was set to 9 ml/win, and the high concentration monomer solution 12b was set to Qml/win, and the flow rates were continuously changed to the dotted line and solid line in FIG. If the length is β, then
It is controlled so that the shallow part is configured. A portion t adjacent to the product unit length E is a portion that is punched out and processed into a sample injection portion. Incidentally, a plan view of the concentration gradient of the coated film is shown in FIG. 2(b), and a side view is shown in FIG. 2 (C1).

In contrast to FIG. 1, a polymerization reaction initiator liquid 3a added to the low concentration monomer liquid 1 and the high concentration monomer liquid 2, respectively,
3b is dissolved and prepared in each tank, and an amount suitable for the monomer liquid is pumped by flow gradient liquid sending pumps 5a, 5b to static mixers 7a, 7b, which are mixing devices, for mixing with the monomer liquid. 7a and 7b are a low concentration monomer solution 1 and a polymerization reaction initiator solution 3a, respectively.

The high-concentration monomer solution 2 and the polymerization reaction initiator solution 3b are mixed to form a low-concentration monomer solution 12a and a high-concentration monomer solution 12b, respectively, and are pumped to the coating head 8.

Details of the coating head portion are shown in Figure 3 (side sectional view).

As shown in FIG. 4 (front cross-sectional view), a low concentration monomer solution 12a and a high concentration monomer solution 12b are placed in the cavity 1, respectively.
3a and 13b, the coating width is expanded to the full extent as shown in FIG. Next, each solution is distributed through distribution pipes 14a and L4, respectively.
b, and is led to a mixing device 16 by a partition plate 15. The mixing device 16 is preferably a static mixer, and a detailed diagram of the static mixer is shown in FIG.
al is a flat honeycomb plate, (bl is a vibrator type spiral (left and right twist). In the mixing device 16, the low concentration monomer solution 12a and the high concentration monomer solution 12b are completely mixed to form a medium liquid for electrophoretic separation. .

The electrophoretic separation medium liquid having a continuous concentration gradient adjusted as described above is guided to the slot opening 18 through the slot 17, and is directed to the web 1 on the coating roller 20.
Coated on top of 9. At this time, back pressure is applied to the differential pressure chamber 21 in order to stabilize the coating.

[For production]

The present invention relates to a method for producing a gradient gel membrane for electrophoresis in which a medium liquid for electrophoretic separation is continuously applied on a continuously running web, and as a method for preparing the medium liquid for electrophoretic separation, First, the low-concentration monomer solution and the high-concentration monomer solution are fed while continuously changing the flow rate ratio, and the corresponding polymerization reaction initiator solution is added and mixed to each solution, and the solution is supplied to each of the coating heads. By supplying the solution to the mouth, the coating head is stably replenished with the low-concentration monomer liquid and the high-concentration monomer liquid at constantly changing flow rate ratios with good reproducibility.

Next, by mixing the two liquids in the coating head using a mixing device, even if there were any foreign substances in the coating liquid that were generated in the cavity, the foreign substances will be completely dissolved and separated. Thanks to the conduit and mixing device, the coating solution has a uniform film thickness distribution in the coating width direction and is applied to the web from the slot-shaped opening, so that the manufacturing method of the present invention ensures uniform coating over the entire coating width. It is possible to produce a gradient gel membrane for electrophoresis that is thick, free of foreign matter, has a continuous concentration gradient in the running direction of the web, has good stability and reproducibility, and has good productivity. .

〔Example〕

Examples of the present invention will be described.

The medium solution for electrophoretic separation is agarose as a low concentration medium: 65g urea 4.200g acrylamide 549g 1.3.5-triacryloyl -hexahydro- 8-triazine 5.7g deionized water
Up to 9.000mj! Make...■Agarose as a high concentration monomer...40g Urea...4,200g Acrylamide...1,830g1.3.5
-Triacryloyl-hexahydro- 3-) Reazine...19g deionized water up to 9.000mJ...
■Trishydroxymethylamine as a buffer solution...121.14g oxalic acid
...65.4g Ethylenediaminetetraacetic acid disodium salt ...7.45g Deionized water
Make up to 1,000mff1... ■Buffer solution■
・・・・・・・・・・・・750m 12.9χ polyvinylpyrroli'ton aqueous solution/Boot@125χ tetramethylethylenediamine liquid...6.7m7! Mixed liquid of... ■ with low concentration monomer ■ 9,000 ml! In addition to each of , high concentration monomer ■9, and OOO Sho β, low concentration monomer solution 1
．． Highly concentrated monomer liquid 2.

Next, as the polymerization reaction initiators 3a and 3b, 2χdi~2ethylhexyl sulfosuccinate liquid...10k j! 0.375
χ Riboflavin phosphate ester sodium salt aqueous solution・150m! 3.75x ammonium teroxonisulfate aqueous solution...160-β is mixed to make it, and the above three liquids are placed in each tank.

The sum of the flow rates of the low-concentration monomer liquid 1 and the high-concentration monomer liquid 2 is set to 19 mβ/win using the flow rate gradient liquid sending pump 4.5, and the flow rates of the polymerization reaction initiator liquids 3a and 3b are adjusted to correspond to the respective polymer liquid volumes With the total as 1.46 mβ/win, the flow rate gradient liquid feeding pump 6a,
At step 6b, the liquid is fed into the static mixer 7a and Tb.

In the static mixers 7a and Tb, the two liquids are mixed and stirred by their own blades to form a low concentration monomer solution 12a and a high concentration monomer solution 12b.

The low-concentration monomer solution and the high-concentration monomer solution are fed using a flow rate gradient liquid pump (4, 5, 6a,
6b) and supplied to the coating head 8 from the respective supply ports.

The changes in the flow rate ratio are shown in Figure 2 (al). Initially, the low-concentration monomer solution 12a starts at 20.46 mj!/min, and the high-concentration monomer solution 12b starts at 2 mβ/l1in, and as time passes, the dotted line and solid line respectively. While changing the flow rate as shown in Figure 2, fbl is the plane of the gel film on the web corresponding to (al). FIG. 3(C) is a side view.

Next, in the present invention, a low concentration polymer solution 12a and a high concentration polymer solution 12b are supplied from separate supply ports in the coating head 8 as shown in FIG. Pour this liquid into each distribution pipe 1.
There is a partition plate 15 between the distribution pipes 14a and 14b, which force-feed both liquids toward the mixing device 16 at 4a and 14b, to determine the wave direction from the distribution pipes.

Low concentration monomer solution 12a combined in mixing device 16
and high-concentration monomer solution 12b are mixed by a self-blading blade in a mixing device that is a static mixer, and become an electrophoretic separation medium liquid that enters the slot 17 and is applied to the web 19 on the coating roller 20 through the slot opening 18. At this time, back pressure is applied to the differential pressure chamber 21 in order to stabilize the coating.

In the present invention, since the fluid resistance of the distribution pipe 14 and the mixing device 16 in the coating head 8 is large, the cavities 13a, 13
The coating liquid in b is uniformly distributed in the width direction, and even if foreign matter is generated due to retention in the cavity, it is completely dissolved in the mixing device, so that the film thickness is reduced over the entire coating width. The distribution was uniform and coating without foreign matter was possible.

〔Effect of the invention〕

The present invention relates to a method for producing a gradient gel membrane for electrophoresis in which a medium liquid for electrophoretic separation is continuously applied onto a continuously running web, and in which the medium liquid for electrophoretic separation is prepared, firstly, a high-concentration liquid is prepared. Continuously change the flow rate ratio of monomer liquid and low concentration monomer liquid to feed them, add and mix the corresponding polymerization reaction initiator liquid to each liquid, and supply the solution to each liquid supply port of the coating head. Then, the two liquids are mixed in a coating head using a mixing device, and the mixed liquid is applied to a web from one slot-shaped opening as a medium liquid for electrophoretic separation. The membrane manufacturing method has made it possible to manufacture gradient gel membranes with stable and reproducible concentration gradients as high-quality products with uniform performance in the width direction with good productivity.

[Brief explanation of drawings]

Fig. 1 is a process diagram of the method for producing a gradient gel membrane for electrophoresis of the present invention, and Fig. 2 is a process diagram for producing a concentration gradient of the present invention (al
is an explanatory diagram of how to change the flow rate ratio, (b) is a plan view of the gel film corresponding to (al), and (C1 is a side view of (b)). Fig. 3 is an embodiment of the coating head of the present invention. side sectional view of
Figure 4 is a front sectional view of Figure 3, Figure 5 is an explanatory diagram of the static mixer of the present invention, (a) is a honeycomb type, (b)
6 is a longitudinal cross-sectional view of a pipe type, and FIG. 6 is a side cross-sectional view of a conventional coating head. 1...Low concentration monomer liquid 2...High concentration monomer liquid 3a, 3b...Polymerization reaction initiator liquid 4.5, 6a, 6b...Flow rate gradient liquid sending pump 7a, 7
b...Static mixer 8...Coating head 9a, 9b...Controller lO...Computer linkage adapter 11...
Personal computer 12a...Low concentration monomer solution 12b...High concentration monomer solution 13a, 13b...Cavities 14a, 14b...Distribution pipe 15...Partition plate 16...Mixing device 17... Slot 18...Slot opening 19...Web 20...Coating roller 21...Differential pressure chamber 21...Coating liquid 23...Cavity 27...
Slot 28...Slot opening 29...Applying head Figure 3 Figure 55! ff (a) (b) 6th
figure

Claims (1)

[Claims] In a method for producing a gradient gel membrane for electrophoresis in which a medium solution for electrophoretic separation is continuously applied onto a continuously running web, the method for preparing the medium solution for electrophoretic separation is first to prepare a high concentration monomer solution. The low-concentration monomer solution is fed by changing the flow rate ratio continuously, the corresponding polymerization reaction initiator solution is added and mixed with each solution, and the solution is supplied to each solution supply port of the coating head. A method for producing a gradient gel membrane for electrophoresis, characterized in that two liquids are mixed in a coating head using a mixing device, and the mixed liquid is applied as a medium liquid for electrophoretic separation onto a web from one slot-shaped opening.