JP2019100775A - Method for producing gel separation ability improver and use of gel separation ability improver - Google Patents

Abstract

To provide a gel separation ability improver used for preparing an electrophoretic gel having a high separation ability.SOLUTION: The method includes at least one refining process of the following (1) and (2): (1) a refining process including a solid-liquid separation step for obtaining supernatant from a galactomannan aqueous solution, and an alcohol precipitation step; and (2) a refining process including a step of adding activated carbon to the galactomannan aqueous solution to remove impurities.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for producing a gel separation ability improver and use of the gel separation ability improver.

  When nucleic acids or proteins are separated according to their size (ie molecular weight), electrophoresis methods using gels such as polyacrylamide gel or agarose gel are mainly performed. In preparing the gel, it is necessary to adjust to a suitable gel concentration according to the separating ability of the gel, and the operation is very complicated. For this reason, a method for easily obtaining an electrophoresis gel having high separation ability is required, and various studies have been made.

  For example, Non-Patent Document 1 discloses a technique of combining agarose with a specific polysaccharide to produce a gel. In addition, Patent Document 1 discloses a technique in which galactomannan irradiated with gamma rays is combined with agarose.

WO 92/12789 (August 6, 1992)

Perlman, D. et. Al., Anal. Biochem. 163, p. 247-254, 1987

  However, the prior art as described above has room for improvement from the viewpoint of easily obtaining an electrophoresis gel having high separation ability. In addition, in preparation of the gel for electrophoresis, it is necessary to adjust to a suitable gel concentration according to the size of the electrophoresis sample to be separated, and the operation is very complicated.

  An aspect of the present invention aims to realize a method for easily producing a gel separation performance improver capable of obtaining a gel for electrophoresis having high separation performance.

In order to solve the above-mentioned problems, as a result of intensive research conducted by the present inventor, it is possible to provide a gel separation ability improver capable of preparing a gel for electrophoresis having high separation ability by a manufacturing method including a specific step. The present invention has been completed. The present invention includes the following aspects.
[1] A method for producing a gel separation ability improver, which comprises one or more of the following purification steps (1) and (2):
(1) solid-liquid separation step of obtaining supernatant from galactomannan aqueous solution;
Subjecting the supernatant to alcohol precipitation to obtain a precipitate; alcohol precipitation step;
(2) A purification step including the step of adding activated carbon to the galactomannan aqueous solution to remove impurities.
[2] The method for producing a gel separation ability improver according to [1], wherein the supernatant is acidic in the alcohol precipitation step.
[3] The method for producing a gel separation ability improver according to [1] or [2], wherein ethanol is used in the alcohol precipitation step.
[4] The method for producing a gel separation ability improver according to any one of [1] to [3], wherein the concentration of the galactomannan aqueous solution is 0.3% to 2.0%.
[5] A gel separation performance improver obtained by the method for producing a gel separation performance improver according to any one of [1] to [4].
[6] A gel for electrophoresis, comprising the agent for improving gel separation ability according to [5] and agar.
[7] An electrophoresis method comprising using the gel for electrophoresis according to [6].
[8] A gel for electrophoresis, comprising the gel separation ability improver according to [5] and agarose.
A gel for electrophoresis according to [9] [8], which comprises using electrophoretic sample containing GelRed ^TM, electrophoresis method.
[10] A gel manufacturing kit for electrophoresis, comprising the gel separation ability improver according to [5].

  According to one aspect of the present invention, there is provided a gel separation ability improver used to prepare an electrophoresis gel having high separation ability.

FIG. 2 is a diagram showing the results of electrophoresis of Example 1 and Comparative Example 1. It is a figure which shows the result of electrophoresis of Example 2 and Comparative Example 2. It is a figure which shows the result of electrophoresis of the reference example 1-the reference example 3. FIG. It is a figure which shows the result of electrophoresis of Example 3, Example 4, Comparative Example 3, and Reference Example 4. It is a figure which shows the result of electrophoresis of Example 5 and Example 6. FIG. It is a figure which shows the result of the electrophoresis of Examples 7-11.

  Hereinafter, although an example of an embodiment of the present invention is described in detail, the present invention is not limited to these. In addition, unless otherwise indicated in this specification, "A-B" showing a numerical range means "A or more, B or less".

  In the present specification, an electrophoresis gel may be simply referred to as "gel". It can be said that the higher the distance between the bands of nucleic acids of different sizes in the gel, the higher the resolution.

[1. Method for producing gel separation ability improver]
The method for producing a gel separation ability improver according to an embodiment of the present invention (also referred to as “production method” in the present specification) comprises the purification step of any one or more of the following (1) and (2): Including:
(1) solid-liquid separation step of obtaining supernatant from galactomannan aqueous solution;
Subjecting the supernatant to alcohol precipitation to obtain a precipitate; alcohol precipitation step;
(2) A purification step including the step of adding activated carbon to the galactomannan aqueous solution to remove impurities.

  The method for producing a gel separation ability improver according to one embodiment of the present invention may include only (1) among the above-mentioned (1) and (2), and may include only (2). It may well contain both (1) and (2). In addition, below, the purification process of said (1) is also called "purification process (1)", and the purification process of said (2) is also called "purification process (2)."

  For example, when DNA electrophoresis is performed using crude galactomannan, DNA bands may not be separated, and smearing of DNA bands may be observed. The reason for this is that (i) the electrophoresis sample contains a substance that competes with the movement of DNA to the anode, and (ii) the presence of a substance having a strong affinity for DNA prevents the movement of DNA. And (iii) the molecular sieve formed by galactomannan and the starting material for electrophoresis gel (agarose etc.) is finer than expected, and transfer of DNA to the anode becomes extremely difficult, etc. The inventor thinks.

  The production method according to one embodiment of the present invention can remove substances that compete with the transfer of nucleic acid to the anode and substances that have strong affinity to DNA. Therefore, the inventors consider that a gel having high separation ability can be produced by using the gel separation ability improving agent obtained by the manufacturing method according to one embodiment of the present invention.

[1-1. Purification process]
The production method according to an embodiment of the present invention includes any one or more of the following purification steps (1) and (2):
(1) solid-liquid separation step of obtaining supernatant from galactomannan aqueous solution;
Subjecting the supernatant to alcohol precipitation to obtain a precipitate; alcohol precipitation step;
(2) A purification step including the step of adding activated carbon to the galactomannan aqueous solution to remove impurities.

  Galactomannan is a polysaccharide having a structure in which D-galactose is linked by α1 → 6 to a linear main chain formed by linking D-mannose by β1 → 4. Galactomannan can be classified into three by the difference in the composition ratio (M / G) of mannose and galactose which comprise. For example, galactomannan derived from locust bean gum is M / G = 4, galactomannan derived from tara gum is M / G = 3, and galactomannan derived from guar gum is M / G = 2 Are known. In embodiments of the present invention, the galactomannan may be any of the three galactomannans described above.

  The galactomannan may be a purified commercially available product. Examples of commercially available galactomannan derived from locust bean gum include Locust bean gum from Ceratonia siliqua seeds (manufactured by Sigma-Aldrich, average molecular weight: 310 kDa) and the like. Examples of commercial products of galactomannan derived from tara gum include SP175 (manufactured by Unitech Foods). As commercially available products of galactomannan derived from guar gum, VIDOCREAM (manufactured by Unitech Foods) may, for example, be mentioned.

  The concentration of galactomannan (that is, the concentration of galactomannan aqueous solution) in 100 wt% of galactomannan aqueous solution is not particularly limited, but 0.3 to 2.0 wt% is preferable, and 1.0 to 1.5 wt% is more preferable. preferable. If the concentration of the galactomannan aqueous solution is within the above range, galactomannan can be suitably dissolved without excessively raising the viscosity of the galactomannan aqueous solution.

<Purification step (1)>
In the production method according to one embodiment of the present invention, the purification step (1) comprises a solid-liquid separation step of obtaining a supernatant from a galactomannan aqueous solution, an alcohol precipitation step of subjecting the supernatant to alcohol precipitation and obtaining a precipitate including.

(Solid-liquid separation process)
The solid-liquid separation step can be performed using a known solid-liquid separation method such as centrifugation, filtration and the like. For example, when the solid-liquid separation method is carried out by centrifugation, the centrifugation conditions (time, temperature, centrifugal force (× g), etc.) depend on the amount of supernatant and the concentration of galactomannan aqueous solution contained in the supernatant, etc. It may be determined accordingly.

  The centrifugation time is preferably 5 minutes to 30 minutes, for example, when centrifugation is performed under the condition of 10000 × g. Moreover, it is preferable that it is 0 degreeC-30 degreeC, and, as for the temperature of centrifugation, it is more preferable that it is 5 degreeC-20 degreeC. If the time and temperature of centrifugation are within the above ranges, the supernatant containing solubles and the precipitate containing insolubles can be separated.

  Also, for example, when the concentration of the galactomannan aqueous solution is 0.3 to 2.0% by weight, the solid-liquid separation method is carried out by filtration using a nylon mesh sheet (for example, product name; nylon mesh sheet 230, TAITEC) It can also be implemented.

(Alcohol precipitation process)
The production method according to an embodiment of the present invention includes the step of subjecting the supernatant obtained by the solid-liquid separation step to alcohol precipitation to obtain a precipitate. In the present specification, this step is also referred to as "alcohol precipitation step".

The alcohol precipitation step may be carried out by a conventional method. The alcohol precipitation step includes, for example, the following steps (i) to (iv).
(I) obtaining an acidic solution by adding an aqueous acetic acid solution to the supernatant;
(Ii) adding an alcohol to the acidic solution obtained by the step (i),
(Iii) centrifuging the solution obtained in step (ii) to recover the resulting precipitate, and
(Iv) A step of drying the precipitate obtained by the step (iv) to obtain a gel separation performance improver.

  In step (i), it is sufficient if the supernatant can be made acidic. Therefore, the amount of the aqueous acetic acid solution may be appropriately determined according to the amount of the supernatant, the concentration of galactomannan contained in the supernatant, and the like. The pH of the above acidic solution is preferably pH 2 to 4. Impurities soluble in acidic solutions are believed to compete with the transfer of DNA to the anode, so it is an objective to remove them.

  In the step (ii), the above-mentioned alcohol is, for example, ethanol and isopropyl alcohol, and ethanol is preferable. The alcohol may be a cooled alcohol having a solution temperature of 5 to 15 ° C., or may be an alcohol at normal temperature. The amount of alcohol may be appropriately determined according to the amount of supernatant, the concentration of galactomannan aqueous solution, the amount of buffer solution, the type of alcohol and the like. For example, in step (i), an aqueous solution of acetic acid may be added to an aqueous solution of galactomannan to obtain an acidic solution, and then ethanol may be added to perform alcohol precipitation. In this case, the weight ratio of the acidic solution to ethanol (= weight of acidic solution: weight of ethanol) is, for example, 0.25: 1 to 4: 1, for example, 0.6: 1.

  In step (iii), centrifugation conditions such as centrifugation time, centrifugation speed and centrifugation temperature may be appropriately determined according to the amount of supernatant and the concentration of galactomannan contained in the supernatant, etc. . For example, the centrifugation time is 5 to 20 minutes, the centrifugal force of centrifugation is 6000 × g to 10000 × g, and the temperature of centrifugation is 5 ° C. to 25 ° C.

  The number of times of repeating step (ii) and step (iii) is not particularly limited, and is, for example, 1 to 2 times.

  Step (iv) is a step of drying the resulting precipitate. The drying method is not particularly limited, and examples thereof include natural drying, air drying, and heat drying. The drying temperature and the drying time may be appropriately determined in accordance with the amount of the obtained precipitate and the like. In the case of heat drying, for example, the drying temperature is 50 ° C to 60 ° C.

<Purification step (2)>
In the production method according to one embodiment of the present invention, the purification step (2) includes the step of adding activated carbon to the galactomannan aqueous solution to remove impurities. Purification step (2) includes, for example, the following steps (I) to (IV).
(I) adding activated carbon to a galactomannan aqueous solution,
(II) leaving the solution obtained in step (I),
(III) centrifuging the solution obtained by the step (II), and
(IV) A step of recovering the supernatant from the solution obtained by the step (III).

  The raw material of activated carbon in step (I) is not particularly limited, and examples thereof include plants such as pine, bamboo and coconut shell, coal, and petroleum. In addition, the form of the activated carbon is not particularly limited, and examples thereof include fibrous, honeycomb, cylindrical, crushed, granular, and powdery. From the viewpoint of efficiently removing the impurities, the powder is preferable. The addition amount of the activated carbon may be appropriately determined according to the concentration and the amount of the galactomannan aqueous solution and the like. By adding activated carbon to the galactomannan aqueous solution, it is considered possible to remove trace substances which are considered to be contained in crude galactomannan and trace substances which affect the separation of nucleic acids.

  In step (II), the time for which the solution obtained by step (I) is allowed to stand is, for example, 10 to 30 minutes.

  Moreover, in step (II), it is preferable to leave the solution while stirring. By stirring, it is thought that adsorption of impurities to activated carbon and solubilization of galactomannan in water can be efficiently performed.

  Step (III) and step (IV) may be repeated multiple times. The number of times of repeating step (III) and step (IV) is not particularly limited as long as activated carbon can be removed, and is, for example, 2 to 3 times. In addition, if necessary, the supernatant may be filtered with a nylon mesh or the like.

  The supernatant obtained in the step (IV) may be used as it is as a gel separation ability improver, or the supernatant may be further freeze-dried or dried and used as a gel separation ability improver. That is, in the production method according to one embodiment of the present invention, the purification step (2) dries the supernatant obtained by the lyophilization step of freeze-drying the supernatant obtained by the step (IV) and the step (IV) A drying step or the like may be included.

[1-2. Dissolution process]
The production method according to an embodiment of the present invention may include the step of obtaining a galactomannan aqueous solution. In the present specification, this step is also referred to as "dissolution step".

  In the dissolution step, galactomannan aqueous solution can be obtained by dissolving galactomannan in water. When dissolving galactomannan, it may be heat-dissolved.

  As heating temperature, what is necessary is just to decide suitably according to the density | concentration of galacto mannan aqueous solution, and 80 degreeC-110 degreeC is preferable. If the heating temperature in the melting step is within this temperature range, galactomannan can be suitably dissolved.

  Heating can be performed using a microwave oven, an autoclave or the like.

  The heating time in the melting step may be appropriately determined according to the concentration of galactomannan, and is, for example, 1 to 2 minutes when using a microwave oven, or 10 to 21 minutes when using an autoclave. If the heating time in the melting step is within this range, galactomannan can be suitably dissolved without excessively raising the viscosity of the galactomannan aqueous solution.

  The galactomannan aqueous solution may be prepared while stirring. The stirring may be performed manually or may be performed by an apparatus equipped with a stirring mechanism. As an apparatus provided with the stirring mechanism, a magnetic stirrer etc. are mentioned.

  Whether or not galactomannan is dissolved in water can be visually confirmed. In the present specification, in the galactomannan aqueous solution, if there is no lump of galactomannan remaining undissolved, it is judged to be uniformly dissolved.

  The dissolution step and the purification step may be performed simultaneously. That is, in the dissolving process for obtaining the galactomannan aqueous solution, the purification process may be performed.

[1-3. Cooling process]
The manufacturing method according to an embodiment of the present invention may include, after the dissolving step, a step of cooling the galactomannan aqueous solution obtained by the dissolving step. In the present specification, this step is also referred to as a "cooling step".

  The cooling time and the cooling temperature of the galactomannan aqueous solution may be appropriately determined in accordance with the amount and concentration of the galactomannan aqueous solution. The galactomannan aqueous solution is preferably allowed to cool overnight, for example. The cooling temperature of the galactomannan aqueous solution is, for example, 3 to 10 ° C.

  The cooling step preferably includes a pre-cooling step of cooling to a constant temperature by leaving the galactomannan aqueous solution to stand. The constant temperature is preferably 20 ° C. to 60 ° C., and more preferably room temperature.

[1-4. Grinding process]
The manufacturing method according to an embodiment of the present invention may include a grinding step of grinding the gel separation performance improver. When the gel separation performance improver is a solid phase, the gel separation performance improver is easily dissolved in the solution by grinding the gel separation performance improver.

[2. Gel separation ability improver]
The gel separation performance improver according to an embodiment of the present invention is manufactured by the manufacturing method according to an embodiment of the present invention. The gel separation performance improver according to one embodiment of the present invention is generally difficult to specify the structure because it uses galactomannan as a raw material for which chemical analysis and identification are not easy.

  The form of the gel separation ability improver according to one embodiment of the present invention is not particularly limited, and may be a solid phase such as powder, or may be a liquid phase.

The gel separation performance improver according to an embodiment of the present invention may be dissolved or mixed in any amount of solvent, as long as the properties are not impaired, and may contain any amount of other components. As a solvent, the below-mentioned buffer solution (Tris-acetic acid-ethylenediaminetetraacetic acid buffer solution and Tris-borate-ethylenediaminetetraacetic acid buffer solution), a tris phosphate buffer, etc. are mentioned. As the other components, ethidium ^bromide, GelRed ^TM, etc. SYBR R Green and DMSO (dimethyl sulfoxide) and the like.

[3. Gel for electrophoresis]
An electrophoresis gel according to an embodiment of the present invention contains a gel separation ability improver according to an embodiment of the present invention. By using the gel separation ability improver according to one embodiment of the present invention, a gel for electrophoresis having high separation ability can be easily prepared.

  The material for electrophoresis gel may be a material generally known as a material for electrophoresis gel such as agar, agarose and polyacrylamide. One of these raw materials for electrophoresis gel may be used alone, or two or more thereof may be used in combination. Agar, agarose, polyacrylamide and the like may be commercially available products. Examples of commercially available agar include purified agar powder (manufactured by Nacalai Tesque, Inc.). Examples of commercially available agarose include Agarose H (manufactured by Nippon Gene) and Agarose S (manufactured by Nippon Gene). Examples of commercially available polyacrylamides include SuperSep Ace (manufactured by Wako Pure Chemical Industries, Ltd.).

  An electrophoresis gel according to an embodiment of the present invention comprises a gel separation ability improver according to an embodiment of the present invention and agar. Moreover, the gel for electrophoresis which concerns on other embodiment of this invention contains the gel separability enhancer and agarose which concern on one Embodiment of this invention. In addition, the gel for electrophoresis according to another embodiment of the present invention may use the gel separation ability improver according to an embodiment of the present invention, and agar and agarose. Although the weight ratio of agarose to the weight ratio of agar is not particularly limited, it is, for example, 2: 1 to 2.5: 1.

Preparation of a gel for electrophoresis according to an embodiment of the present invention is carried out according to an ordinary method referring to, for example, Molecular Cloning: A LABORATORY MANUAL, T. Maniatis, EF Fritsch, J. Sambrook, Cold Spring Harbor Laboratory, 1982. You may The preparation of the electrophoresis gel is performed, for example, by a method including the following steps (α) and (β).
(Α) A step of mixing a raw material for electrophoresis gel and a gel separation performance improver in a buffer solution to obtain a gelation solution,
(Β) a step of gelling the gelation solution to obtain an electrophoresis gel.

  In the step (α), the buffer is preferably a Tris-acetate-ethylenediaminetetraacetate buffer or a Tris-borate-ethylenediaminetetraacetate buffer.

  Tris-acetate-ethylenediaminetetraacetate buffer is also referred to as TAE buffer. TAE buffer may be prepared by dissolving trishydroxymethylaminomethane, acetic acid and ethylenediaminetetraacetic acid (EDTA) in water. Trishydroxymethylaminomethane is also referred to simply as Tris. Tris may be used in the form of a salt, such as Tris-HCl (Tris-HCL). Acetic acid may also be used in the form of acetate (eg, sodium acetate). TAE buffer is preferred from the viewpoint of providing for DNA recovery and genetic engineering techniques.

  Tris-borate-ethylenediaminetetraacetic acid buffer is also referred to as TBE buffer. TBE buffer may be prepared by dissolving trishydroxymethylaminomethane, boric acid and EDTA in water. TBE buffer is preferred from the viewpoint of separating DNA of 1 kbp or less.

  The total concentration of the raw material for electrophoresis gel and the gel separation performance improver in 100% by weight of the gelation solution is preferably 0.4 to 2.0% by weight, and 0.5 to 1.5% by weight It is more preferable that When the concentration is 0.4% by weight or more, the separation ability and strength of the gel can be sufficiently improved. If the said density | concentration is 2.0 weight% or less, the uniform solution for gelatinization can be obtained easily.

  It is preferable that the density | concentration of the raw material of electrophoresis gel in 100 weight% of solutions for gelatinization (namely, density | concentration of the raw material of electrophoresis gel in gel for electrophoresis) is 0.3-1.5 weight%. When the concentration of the raw material of the electrophoresis gel is 0.3% by weight or more, a gel of sufficient strength can be obtained. When the concentration of the raw material for electrophoresis gel is 1.5% by weight or less, a uniform gelation solution can be easily obtained.

  The concentration of the gel separation ability improver in 100% by weight of the solution for gelation may be appropriately determined depending on the concentration of the raw material for electrophoresis gel. For example, when the gel for electrophoresis contains 1.0% by weight of agarose, it is preferably 0.1% by weight or more and less than 0.4% by weight. When the concentration of the gel separation performance improver is 0.1% by weight or more, the separation performance and strength of the gel can be sufficiently improved. If the concentration of the gel separation performance improver is less than 0.4% by weight, a uniform gelation solution can be easily obtained.

  It is preferable to stir the gel separation performance improver and the gel starting material for electrophoresis according to one embodiment of the present invention after being introduced into the buffer solution. The stirring may be performed manually or may be performed by an apparatus equipped with a stirring mechanism. As an apparatus provided with the stirring mechanism, a magnetic stirrer etc. are mentioned.

  In the step (α), it is preferable to dissolve the raw material for electrophoresis gel and the gel separation ability improver in a buffer while heating. Thereby, the raw material for electrophoresis gel and the gel separation performance improver can be easily dissolved in the buffer solution. It is preferable that the heating temperature in a melt | dissolution process is 80-100 degreeC. If the heating temperature is in the above-mentioned range, the raw material for electrophoresis gel and the gel separation ability improver can be sufficiently dissolved. The heating can be performed using a microwave oven or the like usually used in the preparation of a gel for electrophoresis.

  When using a powdery gel separation performance improver in the preparation of a gel for electrophoresis according to one embodiment of the present invention, the raw material of the gel for electrophoresis and the gel separation performance improver are mixed before the step (α). And the step of obtaining a mixed powder. For example, in the case of using agarose as a raw material for electrophoresis gel, the weight ratio of the powder of gel separation ability improver to the powder of agarose in the mixed powder (weight of gel separation ability improver powder / weight of agarose powder) Is preferably 0.3 to 2. When the weight ratio is 0.3 or more, the separation ability and strength of the obtained gel can be further enhanced. In addition, when the weight ratio is 2 or less, the raw material for electrophoresis gel and the gel separation performance improver are easily dissolved in the buffer solution.

  When using a powder of a gel separation ability improver for producing the gel for electrophoresis, the weight ratio may be, for example, 0.8 to 2, or 1 to 2. As described above, even when the concentration of the gel separation performance improving agent is relatively high, the mixed powder is sufficiently dissolved in the buffer solution. Therefore, a gel of sufficient strength can be obtained. This is because the agarose and the gel separation performance improver are mixed in the form of powder. The weight ratio may be 0.3 to 0.6 or 0.3 to 0.5. Thus, even when the concentration of the gel separation performance improving agent is relatively low, the separation performance of the gel can be sufficiently enhanced.

  The method of mixing the powder of agarose and the powder of gel separation performance improving agent is not particularly limited. These powders may be mixed manually, or may be mixed by an apparatus equipped with a stirring mechanism or the like. In any case, it is preferable to sufficiently stir these powders so that the powder of the agarose and the powder of the gel resolution improving agent are uniformly mixed.

  In the step (β), it is preferable to gelate by leaving the gelling solution to stand. The temperature for standing is preferably 20 to 60 ° C., and more preferably room temperature. Here, a gel having a desired shape can be obtained by pouring the gelation solution into a mold and then leaving it to stand. Alternatively, a well may be formed for introducing a sample by inserting a comb into a gelation solution before gelation.

  In the present specification, “gelation” is intended to obtain a gel that does not exhibit fluidity by solidifying the gelling solution. The gel is obtained by gelling a solution in which the above-mentioned gel separation ability improver is dissolved. Therefore, the gel has sufficient strength and high resolution.

[4. Electrophoresis method]
The electrophoresis method according to an embodiment of the present invention is performed using the electrophoresis gel according to an embodiment of the present invention. Since the gel for electrophoresis contains the gel separation ability improver according to one embodiment of the present invention, a nucleic acid (in particular, low molecular weight DNA) is suitable by performing electrophoresis using the gel for electrophoresis. Can be separated.

  The electrophoresis method may be carried out in a routine manner, for example, referring to Molecular Cloning: A LABORATORY MANUAL, T. Maniatis, EF Fritsch, J. Sambrook, Cold Spring Harbor Laboratory (1982).

The staining method of the electrophoresis sample is not particularly limited, but it may be stained by adding a staining reagent to the electrophoresis gel, and shaking the electrophoresis gel after electrophoresis in the staining reagent It may be stained by As the staining reagent, it may be used generally known nucleic acid stain, for example, ethidium bromide, GelRED ^TM (Gene One Corporation) and GelGreen ^TM (Gene One Co., Ltd.).

Also, the electrophoretic method according to another embodiment of the present invention, line using electrophoresis samples containing gel and GelRed ^TM for electrophoresis and a gel resolution enhancer and agarose according to an embodiment of the present invention It will be.

The above-mentioned staining reagents are used for nucleic acid staining applications because many substances emit fluorescence when exposed to ultraviolet light. In addition, the above-mentioned staining reagent intercalates into DNA to make the DNA band clear. Therefore, the DNA band may be easily observed by adding the staining reagent as a DNA intercalating reagent to the electrophoresis sample. As the DNA intercalating reagent, it is preferable to use a staining reagent different from the staining reagent used for staining the electrophoresis gel. For example, after being subjected to electrophoresis samples with the addition of GelRed ^TM electrophoresis, the gel for electrophoresis may be stained with ethidium bromide.

In addition, an electrophoresis gel containing agar (that is, a gel for electrophoresis containing only agar as a raw material of electrophoresis gel, a gel for electrophoresis containing agar and agarose as a raw material of electrophoresis gel) is used to conduct electricity. when performing the electrophoresis, electrophoresis sample may be added GelRed ^TM, may not adding GelRed ^TM electrophoresis sample.

The concentration of GelRed ^TM in the electrophoretic sample 100 wt% (or, GelRed ^TM dilution ratio to be added to the electrophoretic sample) may be determined appropriately depending on the composition of the electrophoresis sample. For example, the loading solution (product name: 6 × Loading Buffer Orange G, manufactured by Nippon Gene Co., Ltd.) with respect to 2 [mu] L, if adding 1μL the GelRed ^TM, GelRed ^TM is preferably being diluted 1000 fold. Dilution of GelRed ^TM can be carried out using DMSO (dimethyl sulfoxide) solution or the like.

[5. Gel production kit for electrophoresis]
An electrophoresis gel production kit according to an embodiment of the present invention includes the gel separation ability improver according to an embodiment of the present invention. Therefore, as described above, a gel having high separation ability can be easily prepared. Hereinafter, the gel production kit for electrophoresis is also simply referred to as "kit". Also, [3. The items already described in [electrophoresis gel] are omitted below, and the above description is used as appropriate.

  The kit may further comprise TAE buffer or TBE buffer as a buffer. The gel separation performance improver according to one embodiment of the present invention can be dissolved in the buffer solution to obtain a gelation solution. A gel can be produced by gelling the gelation solution.

  The buffer may be a stock solution prepared at a higher concentration than the concentration at the time of use. For example, the stock solution may be at a concentration of 5, 10, or 50 times that at which it is used. The stock solution can be diluted and used to make a gel.

  Also, the kit may include a powder of tris-acetic acid-ethylenediaminetetraacetic acid or a powder of tris-boric acid-ethylenediaminetetraacetic acid. These powders can be dissolved in water to obtain TAE buffer or TBE buffer.

  The kit may further include a member for producing a gel, in addition to the gel separation ability improver according to an embodiment of the present invention, the raw material for electrophoresis gel and the buffer solution. For example, the kit may include a tray into which the gelling solution may be poured. The kit may also include a plate for supporting the formed gel. Furthermore, the kit may comprise a comb for forming a well in the gel.

  The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the claims, and embodiments obtained by appropriately combining the technical means disclosed in the different embodiments. Is also included in the technical scope of the present invention.

  Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to the following examples.

[1. Observation of the effect of gel separation ability improver A1 in agar gel]
Example 1
(1) Preparation of gel separation ability improver A1 Gel separation ability improver A1 obtained by the following [a] to [h] was used for electrophoresis.
[A] 1 g of galactomannan derived from locust bean gum (Locust bean gum from Ceratonia siliqua seeds, manufactured by Sigma-Aldrich) is added to 100 mL of purified water, and heated in a microwave oven at 100 ° C. for 2 minutes to obtain galactomannan It was dissolved. It was cooled to room temperature and then left at 5 ° C. overnight.
The solution obtained by [b] [a] was separated into a centrifuge tube and centrifuged at 8000 xg for 10 minutes at room temperature.
The supernatant was separated from the solution obtained by [c] and [b] into a centrifuge tube, and the precipitate was removed. At this time, the removed precipitate was about 0.4 g.
Acidic solution was obtained by adding 20 mL of acetic acid (special grade reagent, manufactured by Wako Pure Chemical Industries, Ltd.) to the supernatant from which the precipitate was removed by [d] [c]. Acidic solution: 100% ethanol was further added to the acid solution so that ethanol = 1: 1, and the tube was capped and shaken quickly 2-3 times up and down. A white fibrous substance appeared immediately upon mixing.
[E] The above centrifuge tube was centrifuged at 8,000 × g for 10 minutes at room temperature, and the obtained precipitate was collected.
[F] 10 mL of ethanol was added to the precipitate and shaken well to remove remaining acetic acid. Then, it was again centrifuged at 8000 × g for 10 minutes at room temperature.
[G] The obtained precipitate was collected and allowed to stand at 60 ° C. for 1 hour to be completely dried.
[H] The dried precipitate obtained above was transferred to a mortar and ground with a pestle.

(2) Dissolution of Agar Agar (purified agar powder, manufactured by Nacalai Tesque, Inc.) 1.0 g and 0.3 g of gel separation performance improver A1 were weighed respectively. These were thoroughly stirred and mixed using a spoon and made into a mixed powder.

  In a 200 mL glass bottle with a lid, 100 mL of TAE buffer (pH 8.0; 40 mM Tris-HCl, 20 mM sodium acetate, 2 mM EDTA) and a stirrer bar were placed. The above mixed powder was put into the glass bottle, covered, and shaken quickly two to three times up and down. The above glass bottle was heated at 100 ° C. for about 2 minutes using a microwave oven to dissolve the agar. Thereafter, the glass bottle was placed on a stirrer and stirred. Thus, a gelation solution was obtained. In the gelation solution, it was confirmed that the agar and gel separation ability improver A1 were completely dissolved. In Example 1, the concentration of the agar and gel resolution improver A1 in 100% by weight of the gelling solution is 1.0% by weight and 0.3% by weight, respectively.

(3) Preparation of gel The sample comb (for 8 wells) and the gel plate (width 54 mm, length 60 mm, height 10 mm) attached to the above-mentioned electrophoresis apparatus were respectively set in the gel preparation tray. 13 mL of the gelation solution was poured into the gel preparation tray. The gelling solution was left to stand at room temperature for 1 hour to gelate.

  A small amount of TAE buffer was placed on the solidified gel and the sample comb was carefully withdrawn. Thus, a gel was produced.

(4) Electrophoresis The gel was set in an electrophoresis apparatus (Mupid mini gel electrophoresis tank, manufactured by Mupid (old advance company)). Desired dilution ratio of ^GelRed TM 1 [mu] L dimethylsulfoxide (DMSO solution diluted by), a loading solution (product name: 6 × Loading Buffer Orange G, manufactured by Nippon Gene) 2μL, 10mM Tris-0.1mM EDTA (pH 8.0) 6. An electrophoresis sample was prepared by adding 5 μL and mixing. The electrophoresis sample of each lane is shown in Table 1. The following (a) or (b) was used as the above-mentioned DNA size marker (described as "marker" in Table 1).
(A) 0.1 to 20 kbp of DNA (Gene Ladder wide 2, manufactured by Nippon Gene Co., Ltd.)
(B) 0.1-2 kbp DNA (Gene Ladder 100, manufactured by Nippon Gene Co., Ltd.)

  10 μL of electrophoresis sample was added to the wells formed in the gel. Electrophoresis was performed at 100 V for 30 minutes at room temperature. As an electrophoresis buffer, TAE buffer (pH 8.0; 40 mM Tris-HCl, 20 mM sodium acetate, 2 mM EDTA) was used. In addition, electrophoresis was implemented by a conventional method with reference to Molecular Cloning: A LABORATORY MANUAL, T. Maniatis, EF Fritsch, J. Sambrook, Cold Spring Harbor Laboratory, 1982.

(5) Staining of gel The gel after electrophoresis was immersed for 15 minutes in a TAE buffer in which ethidium bromide (0.5 μg / mL) was dissolved. Further, the gel was immersed in distilled water for 5 minutes.

(6) Photographing of Gel The gel was photographed using an agarose gel photographing device (UV illuminator; Model BioDoc-It (registered trademark) Imaging system, manufactured by UVP).

Comparative Example 1
A gel was prepared in the same manner as in Example 1 except that the gel separation performance improver A1 was not added in preparation of the gel, and electrophoresis was performed using the gel. In Reference Example 1, the concentration of agar in 100% by weight of the gelling solution is 1.0% by weight.

<Result>
FIG. 1 shows the results of electrophoresis of Example 1 and Comparative Example 1. In FIG. 1, “*” indicates a 2 kbp DNA band based on lane 6. Further, the scale indicating the size of DNA shown in FIG. 1 is based on the band of lane 1 of Example 1. Comparison of the results of Example 1 and the results of Comparative Example 1 shows that when the gel separation ability improver A1 obtained by the purification method (1) is added to the agar gel, the separation ability is particularly high at 100 to 3 kbp. I understand that. Further, from the results of lanes 1 and 2 of Example 1, the electrophoresis using 1% agar gel, the addition of gel resolution enhancing agent A1, without the addition of GelRed ^TM electrophoresed sample, improved gel resolution I know what to do.

  Purified agar is cheaper to obtain than agarose. Therefore, it is suggested that the electrophoresis can be performed more inexpensively by using the gel separation ability improver according to the embodiment of the present invention.

[2. Observation of the effect of gel separation ability improver A1 on agarose gel]
Example 2
A gel was prepared in the same manner as in Example 1 except that 1.0 g of agarose (Agarose S, manufactured by Nippon Gene Co., Ltd.) was used instead of 1.0 g of agar, and electrophoresis was performed using the gel. In Example 2, the concentrations of agarose and gel resolution improver A1 in 100% by weight of the gelling solution are 1.0% by weight and 0.3% by weight, respectively.

Comparative Example 2
A gel was prepared in the same manner as in Example 2 except that the gel separation ability improver A1 was not added, and electrophoresis was performed using the gel. In Comparative Example 2, the concentration of agarose in 100% by weight of the gelling solution is 0.3% by weight.

Reference Example 1
Electrophoresis was performed in the same manner as in Example 1 except that the electrophoresis sample shown in Table 2 was run in lanes 1 to 6, for consideration of the reagent added to the electrophoresis sample. Desired addition reagent dilution (GelRed ^TM is diluted with DMSO solution) 1 [mu] L of loading solution (product name: 6 × Loading Buffer Orange G, manufactured by Nippon Gene) 2μL, 10mM Tris-0.1mM EDTA (pH 8.0 An electrophoresis sample was prepared by adding and mixing 6.5 μL. In Reference Example 1, the concentrations of agarose and gel resolution improver A1 in 100% by weight of the gelling solution are 1.0% by weight and 0.3% by weight, respectively.

Reference Example 2
A gel was prepared in the same manner as in Example 1 except that 0.3 g of galactomannan derived from locust bean was added instead of the gel separation ability improver A1 in preparation of the gel. Thereafter, electrophoresis was performed on the following electrophoresis samples using the gel. Desired addition reagent dilution (GelRed ^TM is diluted with DMSO solution) 1 [mu] L of loading solution (product name: 6 × Loading Buffer Orange G, manufactured by Nippon Gene) 2μL, 10mM Tris-0.1mM EDTA (pH 8.0 An electrophoresis sample was prepared by adding and mixing 6.5 μL. As an addition reagent, SYBR ^R Green (manufactured by Thermo Fisher) and Midori Green Direct (manufactured by Nippon Gene) were used as electrophoresis samples for lane 3. The same DNA size marker as in Example 1 was used. The electrophoresis sample of each lane is shown in Table 3. In Reference Example 2, the concentrations of agarose and galactomannan derived from locust bean in 100% by weight of the gelling solution are 1.0% by weight and 0.3% by weight, respectively.

Reference Example 3
A gel was prepared in the same manner as in Reference Example 2 except that a gel separation ability improver A1 was added instead of galactomannan derived from locust bean, and electrophoresis was performed using the gel. In Reference Example 3, the concentrations of agarose and gel resolution improver A1 in 100% by weight of the gelling solution are 1.0% by weight and 0.3% by weight, respectively.

<Result>
The results of the electrophoresis of Example 2 and Comparative Example 2 are shown in FIG. 2, and the results of the electrophoresis of Reference Example 1 to Reference Example 3 are shown in FIG. In FIGS. 2 and 3, “*” indicates a 2 kbp DNA band based on lane 6.

  From the comparison of the results of Example 2 and Comparative Example 2, it can be seen that the agarose gel to which the gel separation ability improver A1 is added exhibits extremely high separation ability, particularly at 100 bp to 2 kbp. The region of 100 bp to 2 kbp is a region frequently used for analysis of the results of PCR reactions. Therefore, it is suggested that the gel resolution improver according to one embodiment of the present invention may be widely used in the future in the electrophoresis of DNA.

In electrophoresis in which gel separation performance improver A1 was added to agarose, no DNA band appeared and smearing of DNA bands was observed (see lanes 1 and 2 in Example 2). . However, the results of lanes 3-5 of Example 2, by adding in advance the GelRed ^TM electrophoresed sample, DNA bands were found to be separated depending on the capacity of GelRed ^TM. Further, it was found that it is possible that the concentration of GelRed ^TM used in lane 5 of Example 2 to separate most effectively DNA. The concentration of GelRed ^TM used in lane 5 of Example 2 is usually 10 times higher concentrations relative to the amount (one thousand times dilution) to be subjected to in-gel DNA staining.

Further, from the results of Reference Example 1, even if previously added ethidium bromide instead of GelRed ^TM electrophoresed sample, resolution of DNA as compared with the case of not adding ethidium bromide was found not to increase.

Furthermore, from the results of Reference Example 2 and Reference Example 3, even if previously added SYBR ^R Green and Midori Green Direct instead of GelRed ^TM electrophoresis sample, as compared with the case where these not added, resolution of DNA enhancing It turned out not to do. Further, it was found from the result of Reference Example 2 that gel separation ability was not improved even if galactomannan derived from locust bean gum, which is a raw material of gel separation ability improver A1, was added to agarose gel. In the Examples, Comparative Examples and Reference Examples, no significant difference is seen even when the results of Lane 1 and Lane 2 are compared, so DMSO as a solvent does not affect the DNA separation ability. all right.

[3. Observation of the effect of gel separation ability improver A1 on agarose / agar gel]
Example 3
A gel was prepared as in Example 1 except that 0.7 g of agarose and 0.3 g of agar were used instead of 1.0 g of agar. Thereafter, electrophoresis was performed on the following electrophoresis samples using the gel. The electrophoresis sample of each lane is shown in Table 4. Incidentally, the dilution of GelRed ^TM was performed using purified water. In Example 3, the concentrations of agarose, agar and gel resolution improver A1 in 100% by weight of the gelling solution are 0.7% by weight, 0.3% by weight and 0.3% by weight, respectively.

Example 4
A gel is prepared as in Example 3 except that TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM ethylenediaminetetraacetic acid, pH 8.0) is used instead of TAE buffer, and the gel is used Electrophoresis was performed. The electrophoresis sample of each lane is shown in Table 5. In Example 4, the concentrations of agarose, agar and gel separation performance improver A1 in 100% by weight of the gelling solution are 0.7% by weight, 0.3% by weight and 0.3% by weight, respectively.

Comparative Example 3
An example except that 0.6 g of agarose and 0.3 g of agar were used instead of 1.0 g of agar, and 0.3 g of galactomannan derived from locust bean gum was added instead of gel separation ability improver A1 A gel was made as in 1. Electrophoresis was performed using the gel. The electrophoresis sample for each lane is the same as in Example 4. In Comparative Example 3, the concentrations of agarose, agar and galactomannan in 100% by weight of the gelling solution are 0.6% by weight, 0.3% by weight and 0.3% by weight, respectively.

Reference Example 4
A gel was prepared in the same manner as in Example 3 except that the gel separation ability improver was not used, and electrophoresis was performed using the gel. The electrophoresis sample for each lane is the same as in Example 4. In Reference Example 4, the concentration of agarose and agar at 100% by weight of the gelling solution is 0.7% by weight and 0.3% by weight, respectively.

<Result>
FIG. 4 shows the results of electrophoresis of Example 3, Example 4, Comparative Example 3 and Reference Example 4. According to the results of Example 3 and Example 4, also in the agarose / agar gel to which the gel separation ability improving agent is added, the separation ability is particularly high particularly at 100 to 2 kbp, similarly to the agar gel to which the gel separation ability improvement agent is added. It can be seen that Further, from the results of lanes 1 and 2 of Examples 3 and 4, similarly to the agar gel, even electrophoresis using agarose / agar gel, the addition of gel resolution enhancing agent is not previously added GelRed ^TM electrophoresed sample Also, it can be seen that the gel separation ability is improved. Furthermore, it can be seen from the results of Example 5 that even when TBE buffer is used instead of TAE buffer, particularly high resolution of 100 to 2 kbp is exhibited.

  On the other hand, it was found from the results of Comparative Example 3 that gel separation ability was not improved even if galactomannan derived from locust bean gum, which is a raw material of gel separation ability improver A1, was added to agarose / agar gel.

Further, from the results of Reference Example 4, the gel resolution was improved by adding pre-electrophoresis sample GelRed ^TM electrophoresis sample.

[4. Observation of the effect of gel separation ability improver A2 and A3]
Example 5
A gel separation performance improver A2 was prepared in the same manner as in Example 3 except that galactomannan derived from guar gum (VIDOCREAM, manufactured by Unitech Foods Co., Ltd.) was used instead of galactomannan derived from locust bean gum. A gel was prepared in the same manner as in Example 3 except that the gel separation ability improver A2 was used instead of the gel separation ability improver A1, and electrophoresis was performed using the gel. The electrophoresis sample of each lane is shown in Table 6. In Example 5, the concentrations of agarose, agar and gel resolution improver A2 in 100% by weight of the gelling solution are 0.7% by weight, 0.3% by weight and 0.3% by weight, respectively.

Example 6
A gel separation ability improver A3 was prepared in the same manner as in Example 5 except that galactomannan derived from tara gum (SP175, manufactured by Unitech Foods Co., Ltd.) was used instead of galactomannan derived from guar gum. A gel was prepared in the same manner as in Example 5 except that the gel separation ability improver A3 was used instead of the gel separation ability improver A2, and electrophoresis was performed using the gel. The electrophoresis sample for each lane is the same as in Example 5. In Example 6, the concentrations of agarose, agar and gel separation performance improver A3 in 100% by weight of the gelling solution are 0.7% by weight, 0.3% by weight and 0.3% by weight, respectively.

<Result>
FIG. 5 shows the results of electrophoresis of Examples 5 and 6. From the results of Example 5, it can be seen that particularly high gel separation ability is exhibited at 100 to 2 kbp even when gel separation ability improver A2 prepared using guar gum-derived galactomannan is added to the gel for electrophoresis. In addition, according to the results of Example 6, even when gel separation ability improver A3 prepared using galactomannan derived from tara gum is added to the gel for electrophoresis, it shows particularly high separation ability at 100 to 2 kbp. Recognize.

[5. Observation of the effect of gel separation ability improver B1]
Example 7
A gel was prepared using gel separation ability improver B1 and gel separation ability improver B1 according to the following [A] to [H], and electrophoresis was performed.
[A] 1 g of galactomannan derived from locust bean gum (VIDOGUM L175 HQ, manufactured by Unitech Foods Co., Ltd.) is further suspended in 5 mL of ethanol, and then slowly dispersed in 95 mL of water while rotating the stirrer. After stirring for 10 minutes, the galactomannan dispersion was dispensed into two centrifuge tubes.
[B] The solution obtained by [A] was separated into two 50 mL centrifuge tubes. 0.2 g of activated carbon (Wako Pure Chemical Industries, for chromatograms) was added to each centrifuge tube, and left to stand for 30 minutes while rotating using a rotater at 15 rpm at room temperature.
[C] The above centrifuge tube was centrifuged at 10,000 × g for 15 minutes at room temperature.
The supernatant was recovered from the solution obtained by [D] and [C].
[E] The mixture was centrifuged at 10,000 × g for 5 minutes.
[F] Mix 25 mL of the above 1% galactomannan solution, 1 mL of 50 × TAE, and 24 mL of purified water, add 0.35 g of agarose (agarose S, manufactured by Nippon Gene Co., Ltd.) and 0.15 g of agar, and add 100 g with a microwave oven. Heat to dissolve for 2 minutes at ° C. In Example 8, the concentrations of agarose, agar and gel resolution improver B1 in 100% by weight of the gelling solution are 0.7% by weight, 0.3% by weight and 0.5% by weight, respectively.
[G] The gelation solution was poured onto a gel preparation plate and left at room temperature for 50 minutes and at 5 ° C for 10 minutes to gelate, thereby preparing a gel.
[H] 10 μL of electrophoresis sample was added to the wells formed in the gel. Electrophoresis was performed at 100 V for 35 minutes at room temperature. The electrophoresis sample of each lane in Example 7 is shown in Table 7.

  The subsequent steps were the same as “(5) gel staining” and “(6) gel imaging” in <Example 1>.

Example 8
A gel was prepared in the same manner as in Example 7 except that 15 mL of a 1% galactomannan solution was used instead of 25 mL of a 1% galactomannan solution, and electrophoresis was performed using the gel. The electrophoresis sample of each lane is shown in Table 8. In Example 8, the concentrations of agarose, agar and gel resolution improver B1 in 100% by weight of the gelling solution are 0.7% by weight, 0.3% by weight and 0.3% by weight, respectively.

Example 9
A gel separation ability improver B2 was produced in the same manner as in Example 7 except that galactomannan derived from guar gum (VIDOCREAM, manufactured by Unitech Foods Co., Ltd.) was used instead of galactomannan derived from locust bean gum. A gel was prepared in the same manner as in Example 7 except that the gel separation ability improver B2 was used instead of the gel separation ability improver B1. Electrophoresis was performed using the gel. The electrophoresis sample of each lane is shown in Table 9. In addition, preparation of GM dispersion liquid of galactomannan derived from guar gum was performed by melt | dissolving galactomannan derived from guar gum in hot water. In Example 9, the concentrations of agarose, agar and gel resolution improver B2 in 100% by weight of the gelling solution are 0.7% by weight, 0.3% by weight and 0.5% by weight, respectively.

Example 10 and Example 11
A gel separation ability improver B3 was produced in the same manner as in Example 7 except that galactomannan derived from tara gum (SP175, manufactured by Unitech Foods Co., Ltd.) was used instead of galactomannan derived from locust bean gum. A gel was prepared in the same manner as in Example 7 except that the gel separation ability improver B3 was used instead of the gel separation ability improver B1, and electrophoresis was performed using the gel. In Example 10, electrophoresis was performed for 35 minutes, and in Example 11, electrophoresis was performed for 70 minutes. In addition, electrophoresis samples of each lane in Example 10 and Example 11 are shown in Table 10. The marker (c) in the table is Lambda phage / Sty I digest (λ / Sty I) (manufactured by Nippon Gene Co., Ltd.). In Examples 10 and 11, the concentration of agarose, agar and gel resolution improver B3 in 100% by weight of the gelling solution is 0.7% by weight, 0.3% by weight and 0.5% by weight, respectively. .

<Result>
The result of the electrophoresis of Examples 7-11 is shown in FIG. In Example 7 of FIG. 6, “*” indicates a 2 kbp DNA band based on lane 2. Also, in Examples 10 and 11 of FIG. 6, “*” indicates a 2 kbp DNA band based on lane 3. From the results of Example 7 and Example 8, it can be seen that when the gel separation ability improver B1 obtained by the purification method (2) is added to the gel for electrophoresis, particularly high resolution is shown at 100 to 2 kbp. . In addition, from the results of Example 9, it is understood that particularly high resolution of 100 to 2 kbp is exhibited particularly when gel separation ability improver B2 prepared using galactomannan derived from guar gum is added to the gel for electrophoresis. . Furthermore, according to the results of Example 10 and Example 11, when the gel separation ability improver B3 prepared using galactomannan derived from tara gum is added to the gel for electrophoresis, the separation ability is particularly high particularly at 100 to 2 kbp. It is understood that it shows. In addition, according to the result of Example 11, when electrophoresis is performed for 70 minutes using gel for electrophoresis added with gel separation ability improver B3, high separation ability is obtained for DNA of longer chain length from 1000 bp to 20 kbp. It is understood that it shows.

  The present invention can be used in various fields in which analysis involving electrophoresis is performed.

Claims (10)

A method for producing a gel separation ability improver, comprising the purification step of any one or more of the following (1) and (2):
(1) solid-liquid separation step of obtaining supernatant from galactomannan aqueous solution;
Subjecting the supernatant to alcohol precipitation to obtain a precipitate; alcohol precipitation step;
(2) A purification step including the step of adding activated carbon to the galactomannan aqueous solution to remove impurities.   The method for producing a gel separation ability improver according to claim 1, wherein the supernatant is acidic in the alcohol precipitation step.   The method for producing a gel separation ability improver according to claim 1 or 2, wherein ethanol is used in the alcohol precipitation step.   The method according to any one of claims 1 to 3, wherein the concentration of the galactomannan aqueous solution is 0.3% to 2.0%.   The gel separation ability improvement agent obtained by the manufacturing method of the gel separation ability improvement agent of any one of Claims 1-4.   An electrophoresis gel comprising the gel separation performance improver according to claim 5 and agar.   An electrophoresis method comprising using the electrophoresis gel according to claim 6.   An electrophoresis gel comprising the gel resolution improver according to claim 5 and agarose. A gel for electrophoresis according to claim 8, characterized by using an electrophoresis sample containing GelRed ^TM, electrophoresis method.   A gel production kit for electrophoresis, comprising the gel resolution improver according to claim 5.