Glue spreading method for first-layer glue of single-cell gel electrophoresis
Through the dual-stage temperature control method of gradient temperature control solidification and secondary heating stabilization, the problems of uneven gel spreading and unstable coagulation of the first layer of gel in single-cell gel electrophoresis were solved, achieving more efficient DNA damage detection.
Patent Information
- Application Number
- CN202510932039.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-07
- Publication Date
- 2025-09-26
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The existing method for spreading the first layer of gel in single-cell gel electrophoresis has problems such as uneven gel layer, unstable coagulation time, and insufficient adhesion to the glass slide surface, which can lead to cell slippage or gel layer cracking, affecting the accuracy and efficiency of DNA damage detection.
A dual-stage temperature control method of low-temperature curing with gradient temperature control solidification and secondary heating stabilization is adopted, combined with a 50ml beaker as a dissolution container to ensure the uniformity of solution volume and temperature diffusion, form a dense bottom layer through low-temperature curing, and enhance the bonding strength between the adhesive layer and the glass slide during secondary heating.
It significantly improves the uniformity of the gel layer and the stability of the coagulation time, shortens the preparation time, reduces background noise, and improves the success rate of cell fixation and the sensitivity and accuracy of DNA damage detection.
Smart Images
Figure CN120703200A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of single cell gel electrophoresis, and in particular to a method for laying a first layer of gel for single cell gel electrophoresis. Background Art
[0002] Single cell gel electrophoresis (SCGE), also known as the comet assay, was first proposed by Ostling et al. in 1984, and this technology was used to detect double-strand breaks in DNA caused by radiation under neutral conditions. It is an important means of detecting DNA damage, in which the preparation of the first layer of gel directly affects the reliability of cell fixation and subsequent electrophoresis results. In traditional methods, ordinary gel-spreading methods have problems such as uneven gel layer, unstable coagulation time (such as long natural cooling time), and insufficient adhesion to the glass slide surface, which can easily cause cells to slip or the gel layer to crack, affecting the accuracy of the experiment, resulting in low accuracy of DNA damage detection and low experimental efficiency. Although the existing patent involves a gel-spreading device, it does not disclose an optimization solution for the gel-spreading process based on temperature gradient control.
[0003] Based on this, it is urgently necessary for those skilled in the art to propose a method for laying the first layer of gel for single-cell gel electrophoresis, which has a uniform gel layer, stable coagulation time, strong adhesion to the glass slide surface, and improves the accuracy of DNA damage detection and experimental efficiency. Summary of the Invention
[0004] In response to the above technical problems, the present invention provides a method for laying the first layer of gel in single-cell gel electrophoresis, thereby solving the problems existing in the prior art.
[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:
[0006] The present invention provides a method for laying the first layer of gel for single cell gel electrophoresis, comprising the following steps:
[0007] S1. Prepare the gel solution: Prepare 1% normal melting point agarose solution. Weigh the normal melting point agarose solution, place it in a 50 ml beaker, and dissolve it in sterile phosphate buffer. The ratio of the mass of the normal melting point agarose solution to the volume of the sterile phosphate buffer is 1:100. Then, place the beaker in a boiling water bath and heat until it is completely transparent.
[0008] S2, vertical dipping: Dip the pretreated slide vertically and quickly into the agarose solution, so that the agarose solution completely covers the slide surface, avoiding the generation of bubbles, and hold for 5 seconds before removing it at a constant speed;
[0009] S3, gradient temperature control solidification: first perform the initial low-temperature solidification, and then perform secondary heating stabilization after the low-temperature solidification is completed;
[0010] S4, storage for future use: After the slides have been stabilized by secondary heating in S3, they are taken out and placed in a sealed humidified box to prevent dehydration of the adhesive layer.
[0011] Furthermore, in step S1, the 1% normal melting point agarose solution is prepared by mixing 40 ml of PBS and 0.4 g of normal melting point agarose.
[0012] Furthermore, the specific method of pre-treating the glass slide in step S2 is to preheat the glass slide to 50-60° C. in an oven.
[0013] Furthermore, the specific method of the initial low-temperature curing in step S3 is to place the glass slide dipped in S2 horizontally in a 4°C environment for 1 hour to allow the agarose molecules to be oriented and form a dense bottom layer.
[0014] Furthermore, the specific method of the secondary heating stabilization in step S3 is to transfer the slide glass after the initial low-temperature curing to a 37°C constant temperature oven and let it stand for 1 hour to eliminate internal stress and enhance the bonding strength between the adhesive layer and the slide glass.
[0015] Compared with the prior art, the present invention has the following beneficial effects:
[0016] The present invention discloses a method for laying the first layer of gel for single-cell gel electrophoresis. The method adopts dual-stage temperature control of low-temperature solidification of gradient temperature-controlled solidification and secondary heating stabilization, and adopts a 50ml beaker as a dissolution container, thereby ensuring the uniformity of solution volume and temperature diffusion. The method of the present invention can shorten the total cooling time to 2 hours compared with the traditional natural cooling of more than 4 hours, and can avoid the influence of room temperature fluctuations on the quality of gel formation. Compared with the traditional method, the thickness of the gel layer prepared by the method is more uniform, which can reduce the background noise during electrophoresis, and has a higher cell fixation success rate and a stronger experimental reproducibility, thereby significantly improving the sensitivity and accuracy of DNA damage detection. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Attachment Figure 1 This is a flow chart of the method for laying the first layer of gel for single-cell gel electrophoresis in this embodiment;
[0018] Attachment Figure 2 This is a graph showing the significant changes in Tail DNA% between Example 1 and the comparative example;
[0019] Attachment Figure 3 This is the single cell gel electrophoresis result of the comparative example;
[0020] Attachment Figure 4 This is the single cell gel electrophoresis result of Example 2. DETAILED DESCRIPTION
[0021] In order to make the objects and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the examples. It should be understood that the specific embodiments described herein are only intended to explain the present invention and are not intended to limit the present invention. The instruments used in the examples are all commonly used laboratory instruments.
[0022] Example 1
[0023] A method for laying the first layer of gel for single cell gel electrophoresis in this embodiment is shown in the attached Figure 1 , including the following steps,
[0024] S1. Prepare a gel solution: Prepare a 1% normal melting point agarose solution. Weigh the normal melting point agarose solution, place it in a 50 ml beaker, and dissolve it in sterile phosphate buffered saline (PBS). The ratio of the mass of the normal melting point agarose solution to the volume of the sterile phosphate buffer is 1:100. In this example, 0.4 g of normal melting point agarose was weighed and dissolved in 40 ml of sterile phosphate buffer. The beaker was then placed in a boiling water bath and heated until completely transparent.
[0025] S2, vertical dipping: Preheat the slide to 50°C in an oven for pretreatment. Dip the pretreated slide vertically and quickly into the agarose solution, ensuring that the agarose solution completely covers the slide surface. Avoid bubbles. Hold for 5 seconds and then remove at a constant speed.
[0026] S3, gradient temperature control solidification: First, perform the initial low-temperature solidification. Place the glass slide dipped in S2 horizontally in a 4°C environment for 1 hour to orient the agarose molecules and form a dense bottom layer, which can promote their directional gelation. After the low-temperature solidification is completed, perform secondary heating stabilization. Transfer the glass slide after the initial low-temperature solidification to a 37°C constant temperature oven and let it stand for 1 hour to eliminate internal stress and enhance the bonding strength between the glue layer and the glass slide.
[0027] S4, storage for future use: After the slides have been stabilized by secondary heating in S3, they are taken out and placed in a sealed humidified box to prevent dehydration of the adhesive layer.
[0028] Example 2
[0029] A method for laying the first layer of gel for single cell gel electrophoresis in this embodiment is shown in the attached Figure 1 , including the following steps,
[0030] S1, prepare gel solution: prepare 1% normal melting point agarose solution by adding 40 ml PBS and 0.4 g normal melting point agarose. Place the prepared 1% normal melting point agarose solution in a 50 ml beaker, and then heat the beaker in a boiling water bath until completely transparent.
[0031] S2, vertical dipping: Preheat the slide to 55°C in an oven for pretreatment. Dip the pretreated slide vertically and quickly into the agarose solution, ensuring that the agarose solution completely covers the slide surface. Avoid bubbles. Hold for 5 seconds and then remove at a constant speed.
[0032] S3, gradient temperature control solidification: First, perform the initial low-temperature solidification. Place the glass slide dipped in S2 horizontally in a 4°C environment for 1 hour to orient the agarose molecules and form a dense bottom layer, which can promote their directional gelation. After the low-temperature solidification is completed, perform secondary heating stabilization. Transfer the glass slide after the initial low-temperature solidification to a 37°C constant temperature oven and let it stand for 1 hour to eliminate internal stress and enhance the bonding strength between the glue layer and the glass slide.
[0033] S4, storage for future use: After the slides have been stabilized by secondary heating in S3, they are taken out and placed in a sealed humidified box to prevent dehydration of the adhesive layer.
[0034] Example 3
[0035] A method for laying the first layer of gel for single cell gel electrophoresis in this embodiment is shown in the attached Figure 1 , including the following steps,
[0036] S1, prepare gel solution: prepare 1% normal melting point agarose solution by adding 40 ml PBS and 0.4 g normal melting point agarose. Place the prepared 1% normal melting point agarose solution in a 50 ml beaker, and then heat the beaker in a boiling water bath until completely transparent.
[0037] S2, vertical dipping: Preheat the slide to 60°C in an oven for pretreatment. Dip the pretreated slide vertically and quickly into the agarose solution, ensuring that the agarose solution completely covers the slide surface. Avoid bubbles. Hold for 5 seconds and then remove at a constant speed.
[0038] S3, gradient temperature control solidification: First, perform the initial low-temperature solidification. Place the glass slide dipped in S2 horizontally in a 4°C environment for 1 hour to orient the agarose molecules and form a dense bottom layer, which can promote their directional gelation. After the low-temperature solidification is completed, perform secondary heating stabilization. Transfer the glass slide after the initial low-temperature solidification to a 37°C constant temperature oven and let it stand for 1 hour to eliminate internal stress and enhance the bonding strength between the glue layer and the glass slide.
[0039] S4, storage for future use: After the slides have been stabilized by secondary heating in S3, they are taken out and placed in a sealed humidified box to prevent dehydration of the adhesive layer.
[0040] Comparative Example
[0041] This comparative example is a traditional glue laying method, which is as follows:
[0042] Mix the cells to be tested with low-melting-point agarose and spread them on a pre-treated clean glass slide.
[0043] Comet assay was performed on the slides obtained in the above examples and comparative examples, and the Tail DNA % change graph was measured and plotted using CASP and GraphpadPrism software. Figure 2 The slides obtained in Example 2 (after optimization) and the comparative example (not optimized) were electrophoresed (horizontal electrophoresis apparatus, Beijing Liuyi DYCP-31BN) and stained under a microscope. Figure 3 The results of single cell gel electrophoresis using the traditional method (not optimized) in the comparative example are shown in the attached figure. Figure 4 This is the single cell gel electrophoresis result of Example 2 (after optimization).
[0044] By the attached Figure 2 It can be seen that the figure clearly shows that the value of "TailDNA%" in the "optimized" state is significantly increased (from about 7% to nearly 20%) compared to the "unoptimized" state.
[0045] By the attached Figure 3 It can be seen that there are very few red fluorescent spots in the picture, indicating that there are few fixed cells. The only few fluorescent spots are small and isolated. The typical "comet" morphology (i.e., core nuclear DNA and trailing fragmented DNA) is completely invisible. The bright spots are very sparsely distributed and far apart from each other. This shows that the experimental process failed to effectively show DNA damage (for example: insufficient lysis, unoptimized electrophoresis conditions, poor staining effect, etc.). Figure 4 It can be seen that a large number of red fluorescent structures can be seen in the picture, indicating that there are many fixed cells. These structures are clearly displayed as dots and obvious short rods. The dots represent undamaged or slightly damaged cell nuclei (comet heads), and the short rods can clearly show the typical "comet" morphology. The short rods ("comets") usually include a brighter head (core DNA) and an outward-extending, gradually fading tail (migrated fragmented DNA). The structures are relatively densely distributed, occupying most of the area in the picture, clearly detecting extensive DNA damage (represented by the comet tail), and fully showing the typical "comet" morphology, which is a key visual indicator for assessing the type and extent of DNA damage.
[0046] Beneficial effects of this embodiment: The present embodiment provides a method for laying the first layer of gel for single-cell gel electrophoresis, which employs dual-stage temperature control through low-temperature solidification of gradient temperature-controlled solidification and secondary heating stabilization, and uses a 50ml beaker as a dissolution container, thereby ensuring uniformity of solution volume and temperature diffusion. The method of the present invention can shorten the total cooling time to 2 hours compared to the traditional natural cooling of more than 4 hours, while avoiding the influence of room temperature fluctuations on the quality of the gel. Compared with the traditional method, the thickness of the gel layer prepared by this method is more uniform, which can reduce background noise during electrophoresis, increase the success rate of cell fixation, and enhance the experimental reproducibility, thereby significantly improving the sensitivity and accuracy of DNA damage detection.
[0047] The above description is merely a preferred embodiment of the present application and an explanation of the technical principles employed. Those skilled in the art should understand that the scope of the invention involved in this application is not limited to the technical solutions formed by the specific combination of the above-mentioned technical features, but should also cover other technical solutions formed by any combination of the above-mentioned technical features or their equivalents without departing from the inventive concept. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.
Claims
1. A method for laying the first layer of gel for single-cell gel electrophoresis, characterized by: The following steps are included: S1. Prepare the gel solution: Prepare 1% normal melting point agarose solution by weighing the normal melting point agarose solution, placing it in a 50 ml beaker, and dissolving it in sterile phosphate buffer, where the ratio of the mass of the normal melting point agarose solution to the volume of the sterile phosphate buffer is 1:
100. Then, place the beaker in a boiling water bath and heat until it is completely transparent. S2, vertical dipping: dip the pretreated slide vertically and quickly into the agarose solution until the agarose solution completely covers the slide surface, hold for 5 seconds, and then remove at a constant speed; S3, gradient temperature control solidification: first perform the initial low-temperature solidification, and then perform secondary heating stabilization after the low-temperature solidification is completed; S4, storage for future use: After the second heating and stabilization in S3, the slides are taken out and placed in a sealed humidified box.
2. The method for laying the first layer of gel for single cell gel electrophoresis according to claim 1, characterized in that: In step S1, the 1% normal melting point agarose solution is prepared by adding 40 ml of PBS and 0.4 g of normal melting point agarose.
3. The method for laying the first layer of gel for single cell gel electrophoresis according to claim 1, characterized in that: The specific method of pre-treating the glass slide in step S2 is to preheat the glass slide to 50-60° C. in an oven.
4. The method for laying the first layer of gel for single cell gel electrophoresis according to claim 1, characterized in that: The specific method of the initial low-temperature curing in step S3 is to place the glass slide after the dipping in step S2 horizontally in a 4°C environment for 1 hour until a dense bottom layer is formed.
5. The method for laying the first layer of gel for single cell gel electrophoresis according to claim 4, characterized in that: The specific method of the secondary heating stabilization in step S3 is to transfer the glass slide after the initial low-temperature curing to a 37° C. constant temperature oven and let it stand for 1 hour.