JPH01138454A - Production of gradient gel film for electrophoresis - Google Patents

Abstract

PURPOSE:To stabilize product quality by mixing monomer liquids having plural stages of different flow rates and a specified concn. and respective polymn. initiator liquids and coating the liquid mixtures composed of such plural kinds as coating films in horizontal one row on webs from a slot. CONSTITUTION:Medium liquids 6 (6a-6e) for electrophoresis sepn. are obtd. by preparing the monomer liquids 1 (1a-1e) and the polymn. initiator liquids 2 (2a-2e) corresponding to the monomer liquids 1 in respective tanks and force- feeding the respective liquids by means of constant flow rate pumps 3 (3a-3e), 4 (4a-4e) to static mixers 5 (5a-5e), then stirring and polymerizing the liquids in the static mixers. These medium liquids 6 are supplied to the respective liquid feed ports of a coating head 7 by the pressures of the pumps 3, 4, are arrayed in horizontal one row by slit apertures 16 and are coated on the webs 9 on a coating roller 8. The product quality is thus stabilized.

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 method for applying a medium liquid.

[Conventional technology]

Conventionally, in plate electrophoresis, a non-self-supporting acrylamide polymer concentration gradient gel is electrophoresed using a tapered spacer on two supports or between two supports. It has been formed into a tapered shape in the direction and 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,
When storing the gel or adding an analysis sample, it is possible to accidentally break the gel or drop something other than the sample onto the gel, damaging the gel. It was necessary.

On the other hand, in the vertical electrophoresis method, in which a mold is made from two glass plates, etc., gel is formed in the mold, and electrophoretic analysis is performed while the mold is held vertically, the thickness of the mold is made uniform. In addition, the gel forming solution has to be injected into a narrow mold before it becomes a gel, which requires a high degree of operational skill.

In addition, since glass was used, there was a drawback that the glass plate was easily damaged. Further, the film thickness gradient is limited to the taper, and it has been impossible to realize an arbitrary film thickness gradient.

In order to solve the above problems and produce a gradient gel membrane for electrophoresis that has high productivity and high reproducibility, the applicant has developed a method for producing a gradient gel membrane for electrophoresis that has high productivity and high reproducibility. A gradient gel membrane for electrophoresis, in which a medium solution for electrophoretic separation is prepared by mixing a corresponding polymerization reaction initiator solution with a static mixer, and the medium solution for electrophoretic separation is applied onto a continuously running web. proposed a manufacturing method.

[Problem that the invention seeks to solve]

The coating method described above is an excellent method and is suitable for production as a high-grade gradient membrane for electrophoresis with a multi-step film thickness gradient. A method for manufacturing gradient gel membranes with high reproducibility and high productivity has been desired.

・An object of the present invention is to address the above-mentioned problems and provide a method for manufacturing a gradient membrane for electrophoresis for DNA sequencing with a simple several-step concentration gradient with high reproducibility and high 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 liquid for electrophoretic separation is applied onto a continuously running web, in which a monomer liquid of a constant concentration and a monomer liquid of a constant concentration at different flow rates are polymerized in multiple stages. The initiator liquid and the initiator liquid are mixed and prepared, and each of the mixed liquids is separately supplied to multiple liquid supply ports of the coating head in a flow rate layer, and the liquid is spread over the width of each slot, allowing multiple types of flow rates to be applied. This is achieved by a method for producing a gradient gel membrane for electrophoresis containing urea as a denaturing agent, which is characterized in that different mixed solutions are applied as a horizontally aligned coating film onto a web through slot openings.

In the present invention, the continuously running web 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, such as polyethylene terephthalate or bisphenol A. Polyesters like polycarbonate, polymethyl methacrylate, polyethylene.

Vinyl polymers such as polystyrene and polyvinyl chloride,
Polyamides such as nylon, and their copolymers (
Examples, vinylidene chloride, vinyl chloride copolymer) are preferably used.

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 thereof include raw materials such as acrylamide gel, agarose gel, R powder gel, and agar gel. A liquid that becomes 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 modifier is from about 40 wt/v% to about 6%, based on the volume of the aqueous gel containing the monomer and crosslinker.
It is used in the range of 0 wt/v%. When using urea, the amount is from about 6 mol (about 300 g) to a saturated dissolution amount, preferably from about 7 mol (about 420 g) to a saturated dissolution amount, per 1N of an aqueous gel containing a monomer and a crosslinking agent. Can be used within a range.

In the present invention, methods for applying the electrophoretic separation medium include slide bead coating and extrusion coating. Hopper coat, curtain coat, etc. are used. The thickness of the media film to be applied is selected depending on the purpose of separation, but is usually from 50 mm to about 1.0 mm, preferably about 100 mm.
The range is approximately 0.5 strokes from ttm.

The gradient gel membrane for electrophoresis in the present invention refers to one having a stepwise film thickness gradient within one product unit length. In addition, in the present invention, the monomer liquid with different flow rates in multiple stages refers to a monomer liquid having a film thickness of 50 μIm to 1 mm depending on the purpose of use.
The flow rate is changed in multiple stages so that

As a mixing device for each monomer liquid and each polymerization initiator liquid, tank mixing or any other method may be used, but a static mixer can be used as a preferable method. A static mixer is a mixer that stirs the fluid by its own movement when the fluid is passed through the mixer. Examples of tubular types include a spiral tube, an alternating left and right spiral, or a propeller with baffles. be.

Embodiments of the present invention will be described 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. This embodiment will be explained using an example in which the flow rate is divided into five stages.

The electrophoretic separation medium liquid 6 (a-e) is the monomer liquid 1 (
a-e), polymerization reaction initiator liquids 2 (a-e) corresponding to the monomer liquids were adjusted in each tank, and each was pumped by constant flow pumps 3 (a-e) and 4 (a-e). By stirring and mixing in static mixers 5 (ae), electrophoretic separation medium liquids 6 (ae), which are mixed liquids, are prepared.

Five types of liquids 6 (ae) are supplied to pumps 3 (ae).

The liquid is supplied to the liquid supply ports 14 (a-e) of each of the coating heads 7 by a pressure of 4 (a-e), and is applied from the slot openings 16 onto the web 9 on the coating roller 8 in a horizontal line. A differential pressure chamber 10 is provided to apply back pressure to the rear side of the coating liquid bead for stability of coating.

FIG. 2 shows a plan view (a), a side view 0)), and a cross-sectional view (C ).

The mixed liquid 6 (ae) is supplied to the supply ports 14 (ae) of the coating head 7, respectively, and is applied onto the web 9 in one horizontal row from the slot opening 16.

That is, as a gradient gel membrane for electrophoresis, product units are made in the width direction of the web, and the web is processed by a cutting machine.

Figure 3 is a side sectional view of the coating head used in the present invention;
The figure shows a front sectional view of FIG.

In FIGS. 3 and 4, the five types of liquids are pumped 17, respectively.
The supply port 14 (a-e) of the coating head 7 is indicated by (a-e).
). Each method is expanded widthwise within the application head 7 by slots 15 (a-e) without cavities. In this case, the width of each slot is narrow and the spreading angle of the liquid in the slot can be kept at θ≦40'', so the liquid spreads naturally, and there is no accumulation of liquid and the generation of foreign matter due to it. In front of the slot opening 16 Then, the adjacent liquids come into contact with each other, and in the slot opening 16, each layer forms a horizontal line of coating film, which is applied onto the web 9.

[For production]

In a method for producing a gradient gel membrane for electrophoresis in which a medium solution for electrophoretic separation is applied onto a continuously running web, monomer solutions at a constant concentration and each polymerization initiator solution are mixed at different flow rates in multiple stages.・Prepare liquids, supply each mixed liquid to multiple liquid supply ports of the coating hand separately in order of flow rate, spread the liquid across the front width, and apply multiple mixed liquids with different flow rates in a horizontal line. A method for producing a gradient gel membrane for electrophoresis containing urea as a denaturing agent, characterized in that the membrane is applied onto a web through a slot opening; As a gradient film for use, a unit length of the product is formed in the width direction of the web. The mixed liquid at each stage of flow rate is prepared under certain conditions, so the quality is stable, and in the coating head, the coating liquid (mixed liquid) naturally spreads and is applied as a coating film, so it does not stagnate. A coating film of good quality is obtained without the generation of foreign matter, and due to stable manufacturing conditions, the quality and reproducibility of the product are good, and the productivity is also high.

[Example] Regarding one example of the present invention, Sections 1.2, 3. Or, explain using Figure 4.

As electrophoretic separation medium liquid monomer liquid I Ca-e) -1-■ Agarose...... 12.1g Urea...
...840g Acrylamide ...153g1.3.5-)
Liacryloyl-hexahydro-3-triazine...1.62g deionized water up to 1,800mJ! Make and use as a buffer □■ Trishydroxymethylamine ・・・・・・121.14g oxalic acid
・・・・・・・・・65.4g Ethylenediaminetetraacetic acid disodium salt・・・・・・7.45g Deionized water up to 1.000a+1 and make buffer solution■・・
・・・・・・・・・・・・15(1m ii2.9χ polyvinylpyrrolidone aqueous solution・160@425χN, N,
N', N'-tetramethylethylenediamine liquid...
...Add 1.34 ml of mixed liquid -■ to monomer ■1, 800, and add monomer liquid 1 (a-e
).

Next, as the polymerization reaction initiator 2 (a-e), 2Xdi-2 ethylhexyl sulfosuccinate solution...20m10.375χ
Riboflavin phosphate ester sodium salt aqueous solution・30m j! 3.75
χAmmonium peroxodisulfate aqueous solution...32m j! Make a mixed solution of

Next, each monomer solution 1 (a
-e) each 47.5 mj! /min, 9.5mj
! /min+19 mf/+in, 28.5 mj!
/l1lin, 38 +a2/min, and in addition to that, 1.85 ml of each of polymerization reaction initiating solutions 2 (a-e) were added using a constant flow pump 4! ,/win, 0.37
ml.

/ akin, 0.71/! /sin, 1.11
mj! /win, 1.48 tan/1111n+ fi! Then, in the static mixer 5 (ae), the above two liquids are mixed and stirred by self-blading blades, and a mixed liquid, ie, a medium liquid for electrophoretic separation 6 (ae), is produced.

In the static mixer 5 (a-e), the above 2M is mixed and stirred by a self-blading blade, and a mixed liquid, ie, a medium liquid for electrophoretic separation 6 (a-e), is produced.

The electrophoretic separation medium liquid &(ae), which is a mixed liquid, is supplied from each supply port 14 (ae) of the coating pad 7 and applied onto the web 9 through the slot opening 16 .

The application status of the electrophoretic separation medium liquid 6 (a-e) at each flow rate at that time is as shown in Figure 2 (a), and the film thickness of the applied gel film is divided into five stages and is continuous. It will be in a state where it will be coated.

In addition, the product as a media membrane for electrophoretic separation requires a process of curing after coating, a process of punching out a comb as a sample injection port, a process of pasting a cover sheet, a process of cutting the product to length, etc. Only after this process does it become a product.

However, these steps have no relation to the present invention no matter what method or means is used.

〔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 applied onto a continuously running web, and a monomer liquid with a constant concentration and a polymerization reaction initiator at different flow rates in multiple stages. Mix and prepare each liquid, supply each mixed liquid to multiple liquid supply ports of the coating head separately in order of flow rate, spread the liquid to each slot width, and mix multiple types of mixed liquids with different flow rates. The method for producing a gradient gel membrane for electrophoresis containing urea as a denaturing agent is characterized in that it is applied as a horizontally lined coating film onto a web through a slot opening. We were able to manufacture a gradient gel membrane for electrophoresis with good productivity, stable product quality, and good reproducibility.

[Brief explanation of the drawing]

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 an explanatory diagram of the application state of the five types of dark and light electrophoretic separation medium solutions of the present invention. FIG. 3 is a side sectional view of the coating head according to the present invention;
The figure is a front sectional view of FIG. 3. 1 (a-e) ... Monomer liquid 2 (a-e) ... Polymerization reaction initiator liquid 3 (a-e
). 4 (a-e) ... constant flow liquid sending pump 5 (a-e)
... Static mixer 6 (a-e) ...
- Electrophoretic separation medium liquid 7... Coating head 8... Coating roller 9... Web 10... Differential pressure chamber 11...
- Controller 12... Computer linkage adapter 13...
Web 14 (a-e) ... Liquid supply port 15 (a-e) ... Slot 16 ... Slot opening 17 (a-e) ... Metering pump

Claims (2)

[Claims] (1) In a method for producing an electrophoretic gradient gel membrane in which a medium solution for electrophoretic separation is applied onto a continuously running web, a monomer solution with a constant concentration and a polymerization initiator solution with different flow rates in multiple stages are used. The mixed liquids are mixed and prepared respectively, and each of the mixed liquids is supplied to multiple liquid supply ports of the coating head separately in order of flow rate, and the liquid is spread across the width of each slot, and multiple types of mixed liquids with different flow rates are supplied. A method for producing a gradient gel membrane for electrophoresis containing urea as a denaturing agent, characterized in that the membrane is coated onto a web from slot openings in a horizontal row. (2) The thickness change of the gel film made from the plurality of monomer liquids and polymerization reaction initiator liquids with different flow rates is 50 μm or more.
The method for producing a gradient gel membrane for electrophoresis according to claim 1, wherein the membrane has a thickness of 1 mm.