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Gel paving method by using single cell gel electrophoresis

A technology of gel laying and electrophoresis, which is applied in the field of biomolecular cell experiments, can solve the problems of difficult degumming, difficult drying, high degumming rate, etc., and achieves the effect of low cost, easy scoring, and fast glue laying speed

Inactive Publication Date: 2010-10-20
NORTHWEST UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0013] The purpose of the present invention is to provide a simple and fast gel laying method for comet electrophoresis, which is not easy to degumming in the steps of detwisting, cracking, electrophoresis and neutralization, and is easy to dehydrate and dry, which is convenient for film reading and overcomes the existing technical process of gel preparation It is cumbersome, time-consuming, high degumming rate, and not easy to dry.

Method used

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  • Gel paving method by using single cell gel electrophoresis
  • Gel paving method by using single cell gel electrophoresis
  • Gel paving method by using single cell gel electrophoresis

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Embodiment Construction

[0025] specific implementation plan

[0026] Such as figure 1 As shown, the film is close to the cell suspension at an angle of 30-45 degrees, and the film is dragged backward at a constant speed, and the suspension expands along the front end of the film until the cell suspension is spread into a thin film. ~3min. Use glue laying method of the present invention, can spread 5~7 samples ( figure 2 ).

[0027] The present invention will be specifically described below by using the tobacco protoplast as material to perform single-cell gel electrophoresis. 1. Isolation of tobacco protoplasts

[0028] The test material is a sterile seedling of Nicotiana sylvestris. Get fully developed leaves, cut off the leaves, leave the veins, tear off the skin of the veins, cut into sections obliquely, add enzyme solution (2% cellulase Onozuka R-10, 0.5% hemicellulase, 0.5% pectinase Y-23, 0.5% resolase R-10, 0.16mol / L CaCl 2 , 0.1% MES, 0.4mol / L mannitol, pH 5.8-6.2) Enzymolysis in the ...

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Abstract

The invention discloses a gel paving method by using a single cell gel electrophoresis, which comprises the following steps: (1) immersing a clean glass slide in a 0.2-1.0% agarose solution with a constant melting point, and then baking the glass slide at 37 DEG C for further use; (2) uniformly mixing a cell suspension with 1% agarose solution with a low melting point in a volume ratio of 1:1 and dropping the mixed solution onto the coated glass slide; and (3) approaching a gel paving sheet to the cell suspension at an angle of 30-45 degrees, pulling the gel paving sheet backwards at a constant speed, expanding the suspension along the front end of the gel paving sheet, ending the gel paving after the cell suspension is paved to be in a film shape, and solidifying the gel for 2-3 minutes at 4 DEG C. The degumming rate of the gel paving method is reduced to 0, while the gel paving speed is high; furthermore, the method is simple to operate and easy to master and particularly facilitates the large-scale processing on samples.

Description

technical field [0001] The invention relates to a gel laying method for comet electrophoresis, belonging to the technical field of biomolecular cell experiments. Background technique [0002] The degree of DNA damage and the possibility of repair determine the life and death of cells to a certain extent, and its detection method has always been one of the issues that people pay attention to. Although there are many techniques in this area, single-cell gel electrophoresis (SCGE), or comet (comet) electrophoresis, is the most simple, fast, economical and sensitive method. SCGE is the first technology that can effectively detect DNA damage in single cells, and has been applied to the study of DNA damage and repair induced by various nucleated cell managers. [0003] Single-cell gel electrophoresis was invented by Ostling and johanson, and Singh et al. developed it by introducing an alkaline environment on this basis. The DNA strand breaks caused by various physical and chemic...

Claims

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Application Information

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IPC IPC(8): G01N27/447G01N1/28
Inventor 贾敬芬张来军步怀宇
Owner NORTHWEST UNIV
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