Nucleic acid electrophoresis test gel

Abstract

The present invention relates to a nucleic acid electrophoresis test gel, which comprises a liquid agar gel and a solution of the acrylamide methylenebisacrylamide (Acr-Bis). By combining the liquid agar gel with the Acr-Bis solution, the present invention can improve the separation resolution of a gel formed from agar during nucleic acid electrophoresis, thereby making the properties of the nucleic acid electrophoresis test gel similar to those of commercially available agarose gel, and achieving good separation resolution for DNA fragments to be subjected to gel electrophoresis during nucleic acid electrophoresis tests.

Description

[Technical Field]

[0001] The present invention relates to the technical field of nucleic acid electrophoresis, and more particularly to a gel for nucleic acid electrophoresis testing. [Background technology]

[0002] Agarose is a linear neutral polysaccharide consisting mainly of alternating D-galactose and 3,6-anhydro-L-galactose units. It contains a small amount of charged groups such as sulfate and pyruvate, which makes it an ideal electrophoretic medium with low electropermeability and no adsorption to proteins or basic dyes. It is commonly used in nucleic acid electrophoresis tests.

[0003] Agarose-related products are primarily used in medical, scientific research, and biomedical applications, and are closely related to human health. Currently, China relies on imports for most agarose raw materials and related products, due to the high production costs and poor product quality compared to imported products. Therefore, there is an urgent need to develop new products that can replace commercially available agarose electrophoresis media. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention aims to solve at least part of one of the technical problems in the related art. Accordingly, one object of the present invention is to provide a nucleic acid electrophoresis test gel. The nucleic acid electrophoresis test gel produced by the present invention has good separation resolution for DNA fragments to be subjected to gel electrophoresis. [Means for solving the problem]

[0005] In view of the above, according to an embodiment of the present invention, the present invention proposes a gel for nucleic acid electrophoresis testing, which includes an agar gel and a 0.25% by mass to 2% by mass solution of methylenebisacrylamide Acr-Bis, which is an acrylamide.

[0006] The nucleic acid electrophoresis test gel according to the embodiment of the present invention is a gel formed from agar, which is blended with the acrylamide methylenebisacrylamide (Acr-Bis) to achieve improved separation resolution during nucleic acid electrophoresis of the gel formed from agar. As a result, the properties of the nucleic acid electrophoresis test gel are similar to those of commercially available agarose gel, and when performing a nucleic acid electrophoresis test, the separation resolution of DNA fragments to be subjected to gel electrophoresis is good.

[0007] Furthermore, the nucleic acid electrophoresis test gel proposed in the above embodiments of the present invention may further have the following additional technical features.

[0008] Optionally, the agar gel is prepared by weighing 1.50 g of agar strips, adding it to distilled water so that the material to liquid ratio is 1:100 w / v, mixing it uniformly, and treating it in a water bath at 100°C for 5 to 10 minutes.

[0009] Optionally, the Acr-Bis solution is prepared by adding distilled water, buffer, 0.1 mL of 10% ammonium persulfate solution, and 0.004 mL of TEMED to 30% Acr-Bis (29:1) and mixing uniformly.

[0010] Furthermore, the buffer solution is a 1.5 M Tris base solution containing 0.025% to 0.2% SDS and has a pH of 8.0 to 9.0.

[0011] Optionally, the ratio of the agar gel liquid to the Acr-Bis solution is between 5:1 and 40:1.

[0012] Additional aspects and advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the invention. [Brief explanation of the drawings]

[0013] [Figure 1]1 shows the melting temperatures of a nucleic acid electrophoresis test gel according to an embodiment of the present invention, a commercially available agar, and a commercially available agarose. [Figure 2] 1 shows the melting temperatures of a nucleic acid electrophoresis test gel according to an embodiment of the present invention, a commercially available agar, and a commercially available agarose. [Figure 3] 1 shows the solidification temperatures of a nucleic acid electrophoresis test gel according to an embodiment of the present invention, a commercially available agar, and a commercially available agarose. [Figure 4] 1 shows the gel strength of a nucleic acid electrophoresis test gel according to an embodiment of the present invention, a commercially available agar, and a commercially available agarose. [Figure 5] 1 shows the transparency of a nucleic acid electrophoresis test gel according to an embodiment of the present invention, a commercially available agar, and a commercially available agarose. [Figure 6] 1 shows the viscosity of a nucleic acid electrophoresis test gel according to an embodiment of the present invention, a commercially available agar, and a commercially available agarose. [Figure 7] 1 shows the whiteness of a nucleic acid electrophoresis test gel according to an embodiment of the present invention, a commercially available agar, and a commercially available agarose. [Figure 8] 1 is a gel electrophoresis diagram of a nucleic acid electrophoresis test gel according to an embodiment of the present invention, commercially available agar, and commercially available agarose. DETAILED DESCRIPTION OF THE INVENTION

[0014] The technical solutions of the present invention will be described below through specific examples. It should be understood that one or more method steps described in the present invention do not exclude the possibility that other method steps may exist before or after the combination of the steps, or that other method steps may be inserted between these explicitly described steps. It should also be understood that these examples are merely for the purpose of illustrating the present invention and do not limit the scope of the present invention. Furthermore, unless otherwise specified, the numbers of each method step are merely a convenient means for identifying each method step and do not limit the order of each method step or the feasible scope of the present invention. Changes or adjustments to the relative relationships between the steps should be considered within the feasible scope of the present invention if they do not substantially change the technical content.

[0015] In order to better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below. Although exemplary embodiments of the present invention have been described, it should be understood that the present invention can be realized in various forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0016] All of the test materials used in the present invention are common commercial products and can be purchased on the market.

[0017] The present invention will be described below with reference to specific examples, which are merely illustrative and do not limit the present invention in any way. [Example]

[0018] Preparation of agar gel: Weigh out 1.50 g of agar strips and add them to distilled water so that the material to liquid ratio is 1:100 (w / v). Mix evenly. Heat the material in a water bath and stir evenly with a glass rod. Set the water bath temperature to 100°C and time it for 5 minutes. When the time is up, a uniformly dissolved agar gel is obtained.

[0019] Preparation of Acr-Bis solution: Using a pipette gun, weigh out 4.1 mL of distilled water into a beaker, then weigh out 3.3 mL of 30% Acr-Bis (29:1) into a beaker, and then weigh out 2.5 mL of buffer (1.5 M Tris base, 0.1% SDS, pH adjusted to 8.8 with HCl) into a beaker, then add 0.1 mL of 10% ammonium persulfate solution, and finally add 0.004 mL of TEMED. The solution was then mixed uniformly. [Example]

[0020] To the agar gel (16 mL) obtained in Example 1, 4 mL of Acr-Bis solution was added in proportions to prevent solidification of the Acr-Bis solution, thereby obtaining a gel for nucleic acid electrophoresis testing.

[0021] The nucleic acid electrophoresis test gel (modified agar gel) described above was compared with untreated commercially available agar powder (manufacturer: Fujian Yange Marine Biotechnology Co., Ltd.) and commercially available agarose (manufacturer: Shanghai Beijing Biotechnology Co., Ltd.) to measure the nucleic acid electrophoresis resolution and physical and chemical properties (gel strength, turbidity, viscosity) of the above nucleic acid electrophoresis test gel, commercially available agar powder, and commercially available agarose.

[0022] Dissolution temperature: Place 0.1 g of commercially available agar powder, commercially available agarose, or a sample of nucleic acid electrophoresis test gel into a test tube, add 10 mL of distilled water, then stopper the test tube and place it in a 30°C water bath. Raise the temperature at a rate of 1°C / 15 min and observe how the sample dissolves. The temperature at which the solution in the test tube becomes clear and transparent without forming layers is considered the dissolution temperature of the agar.

[0023] As shown in Figure 1, the melting temperatures of commercially available agarose, commercially available agar powder, and nucleic acid electrophoresis test gel are 80.10°C, 88.64°C, and 89.79°C, respectively.

[0024] Melting temperature: A 1.5% by mass agar solution was prepared and heated to dissolve, after which 15 mL was poured into a test tube with an inner diameter of 22 mm. A thermometer was inserted so that the mercury bulb was below the liquid surface, and the tube was placed so that the liquid level was horizontal and allowed to solidify. The next day, a single 3 mm diameter stainless steel bead was placed on the surface of the gel, and the test tube was placed in a water bath and heated, gradually increasing the temperature of the gel. The temperature at which the stainless steel bead suddenly fell to the bottom of the test tube was observed and recorded as the melting temperature.

[0025] As shown in FIG. 2, the melting temperatures of commercially available agarose, commercially available agar powder, and nucleic acid electrophoresis test gel are 90.77° C., 94.36° C., and 91.67° C., respectively.

[0026] Solidification temperature: A 1.5% by mass agar solution was prepared and heated to dissolve, after which 10 mL was poured into a test tube with an inner diameter of 15 mm, and a thermometer was inserted so that the mercury bulb was below the liquid surface. The temperature of the liquid gel was slowly lowered (approximately 1°C / min) until the test tube was tilted 90°, and the temperature at which the liquid surface solidified and no longer flowed was taken as the solidification temperature.

[0027] As shown in FIG. 3, the solidification temperatures of commercially available agarose, commercially available agar powder, and nucleic acid electrophoresis test gel are 37.17° C., 44° C., and 38.7° C., respectively.

[0028] Gel strength: The gel strength of the three agar powder samples is measured according to the national standard GB 1975-2010 agar gel strength measurement method.

[0029] As shown in Figure 4, the gel strength of commercially available agarose, commercially available agar powder, and nucleic acid electrophoresis test gel was 2232 g / cm. 2 , 1189g / cm 2 , and 1075 g / cm 2 is.

[0030] Transparency: Prepare a 1% by weight agar solution, pour it into a cuvette while it is still hot, and leave it at room temperature for 24 h. Scan the sample using a UV spectrophotometer between 400 and 800 nm to determine the maximum absorption wavelength of 700 nm. Measure the transmittance (T) of the sample at this wavelength.

[0031] As shown in Figure 5, the transparency of commercially available agarose, commercially available agar powder, and nucleic acid electrophoresis test gel is 98.9%, 92.4%, and 84.7%, respectively.

[0032] Viscosity: Prepare a 1.5% by mass agar solution, microwave it until the solution becomes homogeneous and transparent, pour it into a 300 mL tall beaker, and heat it in a constant temperature water bath at 60°C for 30 minutes. After the temperature has stabilized, measure the viscosity using a DV-C digital viscometer.

[0033] As shown in Figure 6, the viscosity of commercially available agarose is 4.28 mPa·s, that of commercially available agar powder is 7.38 mPa·s, and that of the nucleic acid electrophoresis test gel is 5.53 mPa·s. Compared to commercially available agar powder, the viscosity of the nucleic acid electrophoresis test gel is significantly lower than that of the commercially available agar powder, indicating improved quality of the agar powder.

[0034] Whiteness: The whiteness of the three agar powder samples is measured according to the national standard GB12097-1989 starch whiteness measurement method.

[0035] As shown in Figure 7, although the whiteness of the gel for nucleic acid electrophoresis tests (68.8%) is lower than that of commercially available agarose (85.59%), there is a significant difference from the whiteness of commercially available agar powder (66.05%), indicating that the whiteness of the gel for nucleic acid electrophoresis tests has been improved to some extent.

[0036] Nucleic Acid Electrophoresis: The above nucleic acid electrophoresis test gel, untreated commercial agar powder, and commercial agarose were poured into an Erlenmeyer flask. The Erlenmeyer flask was placed in a microwave oven. The power and time (1180W, 1-2 min) were adjusted according to the gel concentration and buffer volume to ensure complete dissolution of the gel. Once the gel temperature in the Erlenmeyer flask had cooled to approximately 60°C, nucleic acid fuel (EB ethidium bromide) was added. The liquid gel was poured into a gel tray, and a comb was inserted into the corresponding position. After leaving the gel at room temperature for at least 30 minutes, the comb was removed. The prepared gel, along with the gel tray, was placed in the electrophoresis tank, electrophoresis buffer was poured in, and the marker and nucleic acid to be analyzed were added to the combhole. The electrophoresis conditions were set and electrophoresis was initiated. Upon completion of electrophoresis, the results were analyzed using a gel imaging system or UV spectrophotometer.

[0037] A 1.5% gel was prepared using the nucleic acid electrophoresis test gel described above. A 5000 bp DNA molecular standard was used and run at 120 V for 33 min. As a control experiment, a 1.5% gel was prepared using untreated commercial agar powder and commercial agarose, and electrophoresis was performed. The results are shown in Figure 1. A: Commercial agarose gel; B: Commercial agar powder gel; C: 0.25% polyacrylamide + agar gel; D: 0.5% polyacrylamide + agar gel; E: 1% polyacrylamide + agar gel; and F: 2% polyacrylamide + agar gel. The commercial agarose gel exhibited a dark background, slight impurities, relatively bright bands, and high resolution. The commercial agar gel exhibited a dark background, impurities, relatively dark bands, and poor resolution. Compared with commercially available agarose, the 0.5% nucleic acid electrophoresis test gel was able to separate large nucleic acid molecular fragments more thoroughly, with a dark background color, slight impurities, narrow and bright bands, and high resolution.

[0038] As described above, the analysis of the physical and chemical properties of the nucleic acid electrophoresis test gel showed that the dissolution temperature, gelling temperature, and melting temperature of the nucleic acid electrophoresis test gel were all superior to those of commercially available agarose, and that the viscosity and whiteness were both superior to those of commercially available agar powder and close to those of commercially available agarose.

[0039] In the description herein, a statement referring to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples" means that the specific features, structures, materials, or characteristics described in connection with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, general descriptions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, one skilled in the art may combine or combine different embodiments or examples described herein.

[0040] Although the embodiments of the present invention have been shown and described above, the above embodiments are illustrative and should not be understood as limiting the present invention, and those skilled in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A nucleic acid electrophoresis test gel comprising an agar gel and 0.25% by mass to 2% by mass of a solution of methylenebisacrylamide Acr-Bis, which is an acrylamide.

2. 2. The nucleic acid electrophoresis test gel according to claim 1, wherein the agar gel is prepared by weighing out 1.50 g of agar, adding it to distilled water so that the material to liquid ratio is 1:100 w / v, mixing them uniformly, and treating it in a water bath at 100°C for 5 to 10 minutes.

3. 2. The nucleic acid electrophoresis test gel according to claim 1, wherein the Acr-Bis solution is prepared by adding distilled water, a buffer solution, 0.1 mL of a 10% ammonium persulfate solution, and 0.004 mL of TEMED to 30% Acr-Bis (29:1) and mixing them uniformly.

4. 4. The nucleic acid electrophoresis test gel according to claim 3, wherein the buffer solution is a 1.5 M Tris-based solution containing 0.025% to 0.2% SDS and has a pH of 8.0 to 9.

0.

5. 2. The nucleic acid electrophoresis test gel according to claim 1, wherein the ratio of the agar gel liquid to the Acr-Bis solution is 5:1 to 40:1.

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