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Rapid detection method and kit for single cell DNA damage

A DNA damage and detection method technology, applied in measurement devices, preparation of test samples, instruments, etc., can solve the problems of easy detachment of glue and difficult solution configuration, and achieve the effect of easy degumming, flexible operation, and easier operation in detection.

Inactive Publication Date: 2021-09-17
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention is carried out to solve the above technical problems, improves the current single-cell DNA damage detection system, provides a rapid detection method and kit for single-cell DNA damage, and solves the problems that the glue is easy to fall off and the solution configuration is difficult

Method used

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  • Rapid detection method and kit for single cell DNA damage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Rapid Detection Method of Single Cell DNA Damage

[0035] 1. Laying glue

[0036] Preparation before gel making: Turn on the water baths at 75°C and 37°C half an hour before operation.

[0037] The first layer of glue: the first layer of glue is the bottom glue, choose a smooth glass slide, first wash all the slides, soak them in alcohol for 1 hour, and then dry them with lens cleaning paper; light an alcohol lamp, spread Bake the glass slide on the flame before gluing, add 0.1-0.3mL of agarose gel with common melting point preheated in a 75°C water bath dropwise on one end of the burnt surface, and spread the agarose gel horizontally with a triangular glass rod immediately. Glue it to the other end, and then dry it with the flame of an alcohol lamp, and set it aside.

[0038] The second layer of gel: Take 30-50 μL of cell suspension and 150-250 μL of 1% low-melting point agarose gel and blow and mix them evenly, then take 5 drops of the mixture and drop it ...

Embodiment 2

[0048] Embodiment 2 verification experiment

[0049] 1. Experimental cells

[0050] HepG2 cells were selected as the experimental subjects.

[0051] 2. Test cell preparation and damage treatment

[0052] Take the cells in the logarithmic growth phase to make cell suspension, and the cells are centrifuged to remove trypsin and medium, and then resuspended with fresh medium for use; adjust the cell density to 1×10 5 ~1×10 6 cells / ml (techniqued by a cell counter), inoculated in a 6-well cell culture plate, and after 24 hours, the cells were detected by light microscopy to confirm that the cells adhered to the wall and grew normally, and the medium in the well was discarded, and the drug-infected cells were prepared and continued to culture .

[0053] After diluting the mother solution of the drug into the corresponding concentration (5μM, 10μM, 20μM, 40μM) of the test drug solution with HepG2 cell culture medium, add 2ml of the test drug solution to each well of the prepared...

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Abstract

The invention discloses a rapid detection method and kit for single cell DNA damage. The rapid detection method comprises the following steps: A, spreading gel, namely cleaning an electrophoresis glass slide in 75% ethanol, wiping the electrophoresis glass slide to be dry, spreading a first layer of gel, baking the glass slide once on flame before spreading the gel, dropwise adding 0.1-0.3 mL of common agarose gel at one end of a roasted surface, horizontally spreading and pulling the gel to the other end by using a coating rod, and then drying for later use; then paving a second layer of gel: taking 30-50 microliters of the cell suspension and 150-250 microliters of 1% low-melting-point agarose gel, blowing, beating and uniformly mixing, taking the mixed solution to drop on one side of a glass slide paved with base gel, slowly putting down from one side with gel by using a cover glass, and then removing the cover glass; B, lysing, unwinding, and a preparation method of a cell lysis solution comprising the following steps: weighing 0.12-0.15 g of Tris, 0.8-1.0 g of NaOH, 14.5-15.0 g of NaCl and 3.72-4.0 g of Na2EDTA (Ethylene Diamine Tetraacetic Acid), putting the weighed Tris, NaOH, NaCl and Na2EDTA into a beaker, adding 80mL of deionized water, uniformly stirring with a glass rod to completely dissolve the weighed Tris, NaOH, NaCl and Na2EDTA, adjusting the pH to 10, diluting to to 100ml, storing at 4 DEG C for later use, and adding triton 100 and dimethyl sulfoxide with the final concentration of 1% before use; and C, performing electrophoresis, dyeing and analysis.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a rapid detection method and a kit for single-cell DNA damage. Background technique [0002] With the accelerated development of China's economy, environmental pollution has become increasingly serious. At present, environmental pollution on a global scale is also receiving more and more attention to human health. Among them, DNA damage caused by environmental pollutants will affect the genetic stability of cells, and may have an impact on offspring, causing miscarriage, stillbirth, teratosis and certain genetic diseases in pregnant women. Since cell DNA damage can cause disease and threaten human health, it is very important to detect cell DNA damage and understand whether this damage can cause changes in cellular events. [0003] Since the advent of the alkaline comet experiment, various related laboratories have widely used this technology to detect ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/30G01N1/44G01N1/28G01N1/38G01N21/64
CPCG01N1/30G01N1/44G01N1/28G01N1/38G01N21/6458G01N21/6428G01N2021/6439
Inventor 张阳李萌陶黎明徐文平项光刚朱莲花李瑞瑞王卫国
Owner EAST CHINA UNIV OF SCI & TECH
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