JPS63298048A - Production of gradient gel film for electrophoresis - Google Patents

Abstract

PURPOSE:To obtain a medium liquid for electrophoretic sepn. having good stability and reproducibility by executing the addition of a polymn. reaction initiator liquid in a jet state by using a throttling nozzle at the time of continuously preparing said medium liquid by adding and mixing monomer liquids and polymn. reaction initiator liquid by means of a static mixer. CONSTITUTION:The medium liquid 12 for electrophoresis is fed by stirring and mixing the low-concn. monomer liquid 1, the high-concn. monomer liquid 2 and the polymn. reaction initiator liquid 3 in the static mixer 7 while the flow rate ratios of the prepd. low-/high-concn. monomer liquids 1, 2 are repeatedly changed as shown in the figure. This change is controlled by a controller 9a via a computer linkage adapter 10 in accordance with the command of a personal computer 11. On the other hand, the polymn. reaction initiator liquid 8 is fed at a fixed rate by a constant flow rate liquid feed pump 6 to the static mixer 7 to prepare the medium liquid 12 for electrophoresis. This liquid 12 is coated on a web 13 via a coating head 2. The throttling nozzle is used for a nozzle 17 for adding the polymn. reaction initiator liquid existing in an addition chamber 14 of the static mixer 7 to add the polymn. reaction initiator liquid in the jet state to the medium liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing a gradient gel membrane for electrophoresis for determining the base sequence of DNA or DNA partial decomposition products.

[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
Do not accidentally break the gel or place anything other than the sample on top of the gel when forming a gel on a support, placing it in a 11iJ bath, storing the gel, or adding an analytical sample. There is a risk of dropping the gel and damaging it.
Great care and skill were required in operation.

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 is difficult to do this, and requires a high level of skill in operation, as the gel forming solution must be injected into a narrow mold before it gels.

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 acrylic acid monomer and a crosslinking agent is prepared, and free radicals are generated that absorb light irradiation and initiate polymerization of the monomer. After adding the substances, the solution is formed into the desired gel product, and the monomer solution or dispersion is polymerized and crosslinked by irradiation of the formed solution to change the porosity of the gel. 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.

However, the former method has the disadvantages of high cost due to poor light irradiation equipment and poor productivity, and poor resolution due to the thick gel.3 The reaction initiator remains polymerized in the presence of light even after production.
Stability due to cross-linking reaction.

There are problems such as the inability to obtain reproducible gradient gels, and the latter method is still a punch-type manufacturing method, resulting in poor productivity. It was difficult to branch uniformly, and it was difficult to obtain reproducible gradient gels.

The present applicant has solved the above problems and developed a method for producing a gradient gel membrane for electrophoresis with high productivity and good reproducibility by using two types of monomer liquids, a concentrated and a light one, in which the flow rate ratio is continuously changed. A method for producing a gradient gel membrane for electrophoresis in which a medium liquid for electrophoretic separation obtained by adding a certain amount of a polymerization reaction initiator to a static mixer, mixing and preparing the liquid is retained on a continuously running web. I applied.

[Problem that the invention seeks to solve]

However, it is necessary to continuously mix with a static mixer.
In the products manufactured by continuously applying the prepared electrophoretic separation medium liquid onto a web, unexplained disturbances in the liquid concentration component distribution were sometimes observed.

An object of the present invention is to solve the above-mentioned problems and provide a method for producing a gradient gel membrane for electrophoresis, which has uniform components in an electrophoretic separation medium and has good stability and good reproducibility.

Means for Solving Problem C] As a result of investigating the cause of this, the present inventors found that when adding three liquids to a static mixer, the addition and mixing of the polymerization reaction initiator liquid is affected by the discharge pressure of the monomer liquid. It has been found that the amount of water used affects the components of the electrophoretic separation medium.

That is, an 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 the medium liquid for electrophoretic separation is continuously mixed with a monomer liquid. A method for producing a gradient gel membrane for electrophoresis, characterized in that when adding and mixing a polymerization reaction initiator liquid with a static mixer, the addition of the polymerization reaction initiator liquid is performed in a jet state using a narrowed nozzle. achieved by.

In the present invention, the running web is a sheet-like sheet with good flatness, and may be made of any material as long as it is non-conductive and substantially water-impermeable, such as polyethylene terephthalate or bisphenol A. Polyesters such as polycarbonate, vinyl polymers such as polymethyl methacrylate, polystyrene, and polyvinyl chloride, polyamides such as nylon, and their copolymers (
For example, vinylidene chloride/vinyl chloride copolymer) is preferably used.

The medium liquid for electrophoretic separation in the present invention may be anything as long as it can form a medium membrane for electrophoretic separation, and representative examples thereof include acrylamide gel, agarose gel 9 starch gel, and agar gel.

It is used as a raw material liquid for cellulose acetate porous membranes, filter paper, etc.

As the coating head for the electrophoretic separation medium in the present invention, a coating head such as a slide bead coat, extrusion coat, hombar code, or curtain coat is used.The thickness of the medium film to be coated is selected depending on the purpose of separation. i, Qm is usually from 50 μm, preferably 200
μm to approximately 0.51■ Approximately 70 in the case of porous membranes and filter paper
The range is from μm to about 1fi.

The gradient gel membrane for electrophoresis in the present invention refers to one having a concentration gradient within one gel membrane.

The static mixer in the present invention refers to a mixer that performs stirring action by the movement of the fluid itself when the fluid is passed through the mixer, and examples of the tubular mixer include a spiral tube, an alternating left and right spiral, or a propeller-shaped baffle plate. There are entrances etc.

In the present invention, the electrophoretic separation medium liquid is continuously mixed with a monomer liquid and a polymerization reaction initiator liquid using a static mixer. This term is not limited to mixing and preparing a monomer liquid and a certain amount of a polymerization reaction initiator liquid using a static mixer, but also includes any case where a monomer liquid and a polymerization reaction initiator liquid are mixed.

In the present invention, narrowing down the addition of the polymerization reaction initiator liquid and making it into a jet state using a nozzle means that when adding the polymerization reaction initiator liquid, it will not be affected by the addition pressure of the monomer liquid and will be spouted out. This means that it is added to the monomer liquid while stirring it.

Embodiments of the present invention will be described using figures.

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

The electrophoresis medium liquid 12 consists of a low concentration monomer liquid 1, a high concentration monomer liquid 2. Further, the polymerization reaction initiator liquid 3 is stirred and mixed in a static mixer 7 to form a UIJ liquid.

FIG. 3 is an explanatory diagram of the relationship between the flow rate ratios of two kinds of concentrated and light monomer liquids with different flow rate ratios as one embodiment of the present invention. Continuously changing the flow rate means that, for example, the initially high concentration monomer liquid 2 is Q m l /win.

The low concentration monomer solution 1 starts with a flow rate of 19 ml/min, but if the product unit length of the gradient gel membrane for electrophoresis is l, the high concentration monomer solution 2 is shown as a solid line in the diagram.
The flow rate of the low-concentration Zimmer's solution 1 is changed as shown by the dotted line, and the total flow rate is constant at 19 ml/ll1in, but the concentration changes continuously. The end of product unit length l is 19 m 1 for high concentration monomer liquid 1
/l1lin, low concentration monomer liquid 1 is Omβ/min
This results in a highly concentrated film component. In order to repeatedly change the flow rate ratio in this way, the flow rate gradient liquid pump 4.5 in FIG. 1 is controlled by the controller 9a via the computer linkage adapter 10 in accordance with commands from the personal computer 11.

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 constant amount is sent to the static mixer 7, and the electrophoresis medium liquid 12 is produced by stirring and mixing in the static mixer 7. The electrophoretic medium liquid 12 is supplied to the coating head 8 and will be coated onto the web 13 .

At this time, as shown in FIG. 2, the polymerization reaction initiator liquid is added in a jet state using a narrowing nozzle of the polymerization reaction initiator liquid addition nozzle 17 in the addition chamber 14 of the static mixer 7.

FIG. 4 is a plan view of a gradient gel membrane for electrophoresis prepared using the concentration ratios shown in FIG. 3, and FIG. 5 is a cross-sectional view of FIG. 4.

The switching length t between the product unit lengths β in the figure is used as the sample injection port, and is used as the sample injection port by a cover sheet or the like.

(Function) The present invention provides 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 the medium liquid for electrophoretic separation is continuously mixed with a monomer liquid. When adding and mixing the polymerization reaction initiator liquid with a static mixer, by adding the polymerization reaction initiator liquid in a jet state using a narrow nozzle, the amount of the monomer liquid is larger than the amount of the polymerization reaction initiator liquid. Therefore, in some cases, the discharge of the polymerization reaction initiator liquid is obstructed, causing uneven discharge, resulting in unevenness in the components of the electrophoretic separation medium liquid. A constant amount of liquid is ejected into the monomer liquid without being affected by the monomer liquid pressure, and it further promotes the mixing of the monomer liquid and the polymerization reaction initiator liquid, resulting in stable mixing of the electrophoretic separation medium liquid. Adjustments can now be made.

〔Example〕

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

Medium for electrophoretic separation As a low concentration monomer □■ Agarose......65g Urea...4.200g Acrylamide...549g1.3.5-Triacryloyl- Hexahydro-3-triazine 5.7g deionized water
Up to 9,00On+j! □■ Agarose......40g Urea...4,200g Acrylamide...1,830g1.3.5-
) Liacryloyl-hexahydro-8-triazine...19g Deionized water up to 9. Make OOOmjl' and use it as a buffer solution □■ Trishydroxymethylamine...121.14g oxalic acid
...65.4g Ethylenediaminetetraacetic acid disodium salt ...7.45g Deionized water
up to 1. Make OOOmA and buffer solution...
・・・・・・750n+j! Mixture of 2.9χ polyvinylpyrrolidone aqueous solution, 800Il1125χ tetramethylethylenediamine solution...6.7mβ, low concentration monomer ■9.000ml, high concentration monomer ■9. OOOmj! Add each to each of the following and add each to a low concentration of Zimmer's solution 1. Highly concentrated monomer liquid 2.

Next, as polymerization reaction initiator solution 3, 2χdi-2 ethylhexyl sulfosuccinate solution...100m40.375%
Riboflavin phosphate ester sodium salt aqueous solution・150m 13.752
Ammonium teroxonisulfate aqueous solution...Make a mixed solution of 160mj+ 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 upper monomer liquid 2 is 19 m'j using the flow rate gradient liquid sending pump 4.5! /min,
The flow rate ratio was gradually changed from low concentration to high concentration and from high concentration to low concentration. In addition, the flow rate 1 of the polymerization reaction initiator liquid 3
．． The liquid is fed into the addition chamber 14 of the static mixer 7 at a rate of 46 mf/min using the constant flow liquid feeding pump 6. As shown in FIG. 2, the addition chamber 14 has a low concentration monomer liquid addition nozzle 1.
5. There were a high-concentration monomer liquid addition nozzle 161 and a polymerization reaction initiator liquid addition nozzle 17, and the polymerization reaction initiator liquid addition nozzle 17 was a narrowing opening, and 1.46 mR of liquid was constantly discharged in a jet state.

In the static mixer 7, the above three liquids are mixed and stirred by self-blading blades to form a medium for electrophoretic separation. You can do ft12.

The electrophoretic separation medium liquid 12 is applied to the web 1 from the coating head 8.
3 is coated on top.

At that time, the flow rate ratio between the lower concentration Upper Zummer's solution and the higher concentration Upper Zummer's solution is as shown in FIG. 2, and the concentration of the applied gel film is as shown in FIGS. 3 and 4.

In addition, the product as a medium membrane for electrophoretic separation is manufactured by a process of pasting spacers on both ends of the web in advance, as disclosed in, for example, Japanese Patent Application Laid-Open No. 60-203847.
A process of curing after application.

It becomes a product only after going through the process of punching out a comb as a sample injection port, the process of attaching a cover sheet, and the second process of cutting the product to length.

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 for electrophoretic separation is coated on a continuously running web, and a method for producing a gradient gel membrane for electrophoresis. When the medium liquid is continuously added and mixed with the monomer liquid and the polymerization reaction initiator liquid using a static mixer, the addition of the polymerization reaction initiator liquid is performed in a jet state using a narrowed nozzle. The method for manufacturing gradient gel membranes for electrophoresis eliminates the turbulence of the conventional medium liquid components for electrophoresis, making it possible to manufacture gradient membranes for electrophoresis with uniform liquid components and good stability and 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, Fig. 2 is a side view of the addition chamber of a static mixer explaining the addition situation of the polymerization reaction initiator liquid of the present invention, and Fig. 3 The figure is an explanatory diagram of the product and the relationship between the flow rate ratios of two kinds of concentrated and light monomer liquids with different flow rate ratios as an example of the present invention. FIG. 4 is a plan view of a gradient gel membrane for electrophoresis of the present invention prepared using the concentration ratios shown in FIG. 3, and FIG. 5 is a cross-sectional view of FIG. 4. 1...Low concentration upper Zimmer solution 2...High concentration monomer solution 3...Polymerization reaction initiation agent solution 4.5...Flow rate gradient liquid feed pump 6...Constant flow rate liquid feed pump 7... -Static mixer 8...Coating heads 9a, 9b...Controller 10...Computer linkage adapter 11...
Personal computer 12... Electrophoretic separation medium liquid 13... Web 14... Addition chamber 15... Low concentration monomer liquid addition nozzle 16... High concentration monomer liquid addition nozzle 17... Start of polymerization reaction Chemical solution addition nozzle procedure amendment 1. 1986 Patent Application No. IJ+ 50b 2, Title of Invention Method for Manufacturing Gradient Gel Membrane for Electrophoresis 3, Person Making Amendment Relationship to Case: Name of Patent Applicant (520) Fuji Photo Film Co., Ltd. 4, Agent 〒100 8, Contents of amendment (1) Figure 1 will be replaced with the attached one. (2) The "Detailed Description of the Invention" column of the specification shall be amended as follows. 1) "Manufacturing method." on page 3, line 13 of the specification cylinder is corrected to "manufacturing method." 2) "Manufacturing method." on page 3, line 20 of the same book is amended to read "manufacturing method." 3) "Propeller-shaped baffle plate included" on page 7, line 16 of the same book is corrected to "propeller-shaped baffle plate included." 4) "Monomer liquid 1" on page 9, lines 8-9 of the same book is corrected to "monomer liquid 2." 5) After "25%" on page 12, line 19 of the same book cylinder, rN, N
, N', N'-J. 6) On page 13, line 10 of the same book, "chiloxo" is corrected to "peroxo". 7) "Mixed solution" on page 13, lines 11-12 of the same book is corrected to "mixed solution." 8) Change r 4.5J from page 13, line 13 of the same book to r 4.5
Correct it with J. 9) “Liquid” after “separation medium” on page 15, line 5 of the same book
Insert. 10) Delete "in the method for producing a gradient gel membrane for electrophoretic separation" from lines 6 to 7 on page 15 of the same book.

Claims (1)

[Claims] In 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, the medium liquid for electrophoretic separation is continuously mixed with a monomer liquid and a polymerization reaction initiator liquid. A method for producing a gradient gel membrane for electrophoresis, characterized in that when adding and mixing with a static mixer, a polymerization reaction initiator solution is added in a jet state using a narrowed nozzle.