JP3633730B2 - Method for forming electrophoresis gel and preparation for electrophoresis gel - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for forming an electrophoretic gel used as a support in the separation and purification of DNA, RNA, protein, and the like, and an electrophoretic gel preparation.
[0002]
[Prior art]
Conventionally, agarose gel and polyacrylamide gel have been used as a support for electrophoresis of DNA or the like. Agarose is a main component of agar extracted from seaweed such as red algae, and is obtained by purifying agar. Polyacrylamide is made purely chemically by polymerizing acrylamide and the cross-linking agent methylenebisacrylamide. Usually, these are properly used according to the size of DNA to be subjected to electrophoresis. Polyacrylamide is suitable for separating DNA of 10 to 1000 bp, and agarose gel is suitable for separating DNA of 100 to 100,000 bp.
[0003]
Agarose is a base material that is easy to handle and easy to use because it is extracted and purified from seaweed, but has the following disadvantages as an electrophoresis gel. First, there is a limit to resolution, and it is impossible to fractionate low molecular weight DNA like polyacrylamide gel. Second, the transparency is not high and it is cloudy, which makes analysis such as determination of base sequence difficult. In particular, when a high-concentration gel is used to fractionate low-molecular DNA, the cloudiness increases. Specifically, in order to confirm the state after electrophoresis, DNA is stained with a fluorescent dye ethidium bromide and irradiated with an ultraviolet lamp to develop a fluorescent color. However, the background is irregularly reflected during photography. Therefore, it is difficult to obtain a clear base sequence image. Third, it contains an anionic group such as a sulfate group as a trace amount of impurities, which causes an electroosmotic phenomenon by electrophoresis. This is a big problem particularly in isoelectric focusing.
[0004]
In order to solve the problems of the agarose electrophoresis gel described above, the following method has been proposed.
(1) A highly refined agarose developed for low molecular weight separation, which is hydroxyethylated to make the network structure finer when agarose is used at a high concentration.
(2) A method of increasing the transparency of a gel and introducing a fine network structure by mixing agarose and γ-irradiated galactoglucomannan (see Japanese Patent Publication No. 7-86503).
[0005]
[Problems to be solved by the invention]
However, since the method (1) requires a reaction step, there is a disadvantage that the cost is high. In addition, in any of the methods (1) and (2), the third problem, that is, the problem of high electroosmosis (EEO) cannot be solved when applied to the isoelectric focusing method.
[0006]
An object of the present invention is to provide a method for forming an agarose electrophoresis gel and a preparation for an electrophoresis gel that eliminate the above-mentioned drawbacks, exhibit high transparency, low electroosmosis, and excellent resolution.
[0007]
[Means for Solving the Problems]
The method for forming an electrophoretic gel according to the present invention comprises adding tamarind gum extracted and purified from tamarind seeds together with agarose extracted and purified from agar to an electrophoresis buffer, and heating to form a transparent sol. It is characterized by forming an electrophoresis gel by casting cooling.
In the present invention, agarose and tamarind gum are preferably added at a ratio of 5 to 80% by weight of tamarind gum with respect to their total amount.
The present invention is also an electrophoretic gel preparation for forming an electrophoretic gel by casting and cooling the transparent sol by forming a transparent sol by heating in an electrophoretic buffer and dissolving the agar. It is characterized in that it contains 20 to 95% by weight of agarose extracted and purified, and the remainder is tamarind gum extracted and purified from tamarind seeds.
[0008]
Tamarind gum is a low-viscosity natural polysaccharide gum extracted from the seeds of the perennial leguminous plant Tamarind growing in Southeast Asia. It has excellent transparency, Newtonian fluid viscosity, and heat resistance is high in many other gums. High compared to quality.
When this tamarind gum is combined with agarose to form an electrophoretic gel, an electrophoretic gel having a finer network structure and less white turbidity than that of agarose alone can be obtained. Moreover, the electroosmosis degree becomes low by adding tamarind.
[0009]
The reason why white turbidity is reduced by adding tamarind is considered as follows. Agarose takes the molecular state of a random coil in solution and takes a helix structure upon cooling. At this time, by strongly attracting water molecules bonded to the helix, the refractive index is increased and the light transmittance is lowered. On the other hand, when tamarind gum enters, the deviation of the above-mentioned combined water decreases, and a decrease in light transmittance is suppressed.
In addition, the reason why the electroosmosis is lowered by adding tamarind gum is that the viscosity of the gel increases when the tamarind gum enters the gel, and the electric double layer on the contact surface with the buffer relaxes the ionization group, thereby supporting it. This is thought to weaken the charge of the body.
Therefore, according to the present invention, there is an advantage that it can be applied particularly to isoelectric focusing.
[0010]
In this invention, when the ratio of tamarind gum is less than 5% by weight with respect to the total amount of agarose and tamarind gum, the effects of improving transparency and reducing electroosmosis cannot be obtained sufficiently. On the other hand, if the tamarind gum exceeds 80% by weight, the ability to form an agarose gel is lowered, the workability is deteriorated, and it is difficult to obtain a uniform gel.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
Examples of the present invention will be described below.
Gel tray in which 2 wt% agarose and 0.5 wt% purified tamarind gum are heated and dissolved in electrophoresis buffer TAE (Tris-acetate containing EDTA) and then slot forming comb is set And solidified by cooling.
Electrophoresis was performed at a constant voltage of 50 V for 2 hours using DNA molecular weight markers φx174 / Hae III digest and pBR322 / Msp I digest.
[0012]
As a comparative example, a gel mixed with agarose and γ-irradiated galactoglutmannan was electrophoresed under the same conditions.
The samples of Examples and Comparative Examples were stained with ethidium bromide, and then the DNA pattern was observed with a transilluminator.
FIG. 1 is a photograph of the patterns of the example and the comparative example. It was confirmed that the gels of the examples were clearly superior in resolution and a clear pattern was obtained.
[0013]
In addition, a preparation in which purified agarose powder and tamarind gum powder are uniformly mixed at a ratio of 5 to 80% by weight of the tamarind gum is prepared, and an electrophoresis gel is similarly formed using this preparation. As a result of the electrophoresis test, it was confirmed that a significant difference was obtained in terms of resolution as compared with the case of agarose alone.
[0014]
【The invention's effect】
As described above, according to the present invention, agarose and tamarind gum are placed in an electrophoresis buffer and heated to form a transparent sol. Thus, electrophoresis has low electroosmosis, is transparent, and exhibits excellent resolution. A gel is obtained.
[Brief description of the drawings]
FIG. 1 is a photograph showing an electrophoresis pattern using gels of Examples and Comparative Examples.

Claims (3)

Tamarind gum extracted and purified from tamarind seeds is added to electrophoresis buffer together with agarose extracted and purified from agar, heated to form a transparent sol, and this transparent sol is cast and cooled to form an electrophoresis gel. A method for forming an electrophoretic gel. The method for forming an electrophoretic gel according to claim 1, wherein agarose and tamarind gum are added at a ratio of 5 to 80% by weight of tamarind gum with respect to the total amount thereof. It is a preparation for forming a transparent sol by heating and dissolving in an electrophoresis buffer, and casting and cooling the transparent sol to form an electrophoresis gel. Agarose extracted and purified from agar is 20-95. An electrophoretic gel preparation characterized by comprising, by weight, tamarind gum extracted and purified from tamarind seeds.

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