CN100573131C - A kind of sample preparation methods and kit that is used for the single cell gel electrophoresis graphical analysis - Google Patents
A kind of sample preparation methods and kit that is used for the single cell gel electrophoresis graphical analysis Download PDFInfo
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- CN100573131C CN100573131C CNB2006101230523A CN200610123052A CN100573131C CN 100573131 C CN100573131 C CN 100573131C CN B2006101230523 A CNB2006101230523 A CN B2006101230523A CN 200610123052 A CN200610123052 A CN 200610123052A CN 100573131 C CN100573131 C CN 100573131C
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Abstract
The invention discloses a kind of sample preparation methods that is used for the single cell gel electrophoresis graphical analysis, comprise shop glue, lysis, untwist wash with electrophoresis, neutralization, the fluorescent dye step, the gel of wherein said shop glue system that step is spread is two-layer; It is the SGD dye solution that described fluorescent dye step adopts asymmetric cyanine class dyestuff, and described SGD dye solution is the solution that SYBR Gold dyestuff is diluted with 1: 10000 volume ratio with dimethyl sulfoxide (DMSO).The present invention also provides a kind of kit of implementing above-mentioned sample preparation methods that is specifically designed to, and comprises 7 kinds of reagent that configure and 6 frosted microslides of handling well.The present invention adopts the SGD dye solution to carry out fluorescent dye, and is nontoxic, and the experimental waste of generation does not pollute the environment, and has higher fluorescence sensitivity; The gel of the system of spreading has only two-layer glue, compares with the technology that traditional sample slide will be spread three layers of gel, and specimen preparation technology is simple, easy operating, success ratio height.
Description
Technical field
The present invention relates to the cell detection technology, particularly a kind of kit that is used for the sample preparation methods of single cell gel electrophoresis graphical analysis and is used to implement this method.
Background technology
Single cell gel electrophoresis graphical analysis (single cell gel electrophoresis, SCGE) technology, claiming comet detection method (comet assay) again, is simple, sensitive, the new technology fast of the research that grows up a kind of end of the eighties and detection by quantitative individual cells dna damage.Bring out down in the relevant factor, the DNA of cell can cause that its higher structure changes, and causes its superhelix loose if sustain damage.When phenomenons such as the cracking of cell generation original position, DNA untwist, the dna fragmentation of damage can shift out from nuclear when electrophoresis, and the anode migration also presents the distribution of tail shape, and the harmless part of cell still keeps spherical.Behind fluorescent dyeing, available fluorescence microscope forms the comet formation pattern to these cells; And under the certain condition, the distribution of the migration content (showing as fluorescence intensity) of the migration distance of DNA (showing as comet tail length) and DNA is relevant with the dna damage degree, and this is the basis of the research and the quantitative test of single cell gel electrophoresis image analysis technology.
Method in common is foundation and improved alkaline single cell gel electrophoresis graphical analysis (alkaline single cell gel electrophoresis such as employing Singh NP in the world at present, ASCGE) technology, on the one hand under high pH value alkali condition, alkali changeableness position and chain rupture more are prone in the cell DNA, also can impel the RNA degraded, so the sensitivity that detects is higher; On the other hand, utilize alkali condition dissolved cell and electrophoresis, improved the susceptibility that detects; In addition, can detect dna double bound rupture and single-strand break simultaneously, and determine susceptibility loci on the DNA chain, thereby make this The Application of Technology scope extremely extensive by adding specific DNA restriction endonuclease.With other DNA bound rupture detection technique, compare as hybridization in situ technique, high performance capillary electrophoresis technology, high performance liquid chromatography, gas chromatography-mass spectrography etc., the single cell gel electrophoresis image analysis technology has characteristics such as sensitivity, quick, easy, good reproducibility, low consumption.Thereby, the single cell gel electrophoresis graphical analysis becomes popular DNA infringement detection method rapidly, be widely used in genetic toxicology, occupation and environmental organism monitoring, radiobiology, oncology, gerontology, Environmental ecology and with oxidative stress and apoptosis-related multiple pathologic process research; And can carry out medicine to the inhibition of dna damage with repair check, tumor radiotherapy chemotherapeutic efficacy assessment check, the damage check of radiation pair cell DNA, the dna damage check of viral pair cell, qualitative, quantitative diagnosis and the efficacy analysis and the public security forensic toxicological analysis etc. of disease.
The specimen preparation basic step that is used for the single cell gel electrophoresis graphical analysis comprises: the shop glue of (1) sample gel slide; (2) lysis; (3) untwist and electrophoresis; (4) neutralization washing; (5) fluorescent dye.The sample slide for preparing just can utilize the single cell gel electrophoresis image analysis system to shoot with video-corder the fluoroscopic image of analysis of cells, and the degree of injury of DNA is estimated.The sample slide of single cell gel electrophoresis graphical analysis for a long time generally all is to adopt ethidium bromide (Ethidium Bromide, EB) carry out fluorescent dye as nucleic acid dye, but the strong carcinogenicity of EB not only threatens user's health, and experimental waste is difficult to again handle contaminated environment.In addition, the sample slide of existing single cell gel electrophoresis graphical analysis will be spread three layers of gel, and preparation technology is complicated, and other preparation process is also comparatively loaded down with trivial details, causes usually to be difficult to prepare suitable sample, has also influenced the popularization and the application of this method.
Summary of the invention
The objective of the invention is to overcome the shortcoming that exists in the prior art, a kind of nontoxic, safe, highly sensitive, sample preparation methods that technology simply is used for the single cell gel electrophoresis graphical analysis is provided.
Another object of the present invention is to provide a kind of kit that is used to implement said method.
Purpose of the present invention is achieved through the following technical solutions:
A kind of sample preparation methods that is used for the single cell gel electrophoresis graphical analysis, comprise shop glue, lysis, untwist wash with electrophoresis, neutralization, the fluorescent dye step; The gel of wherein said shop glue system that step is spread is two-layer; Described fluorescent dye step adopts asymmetric cyanine class dye solution (SGD dye solution).Described asymmetric cyanine class dye solution (SGD dye solution) is the solution that asymmetric cyanine dye SYBRGold is diluted with 1: 10000 volume ratio with dimethyl sulfoxide (DMSO) (DMSO).
Described shop glue step, be earlier with the frosted microslide 38～42 ℃ of preheatings, then 1% normal fusing point Ago-Gel evenly is coated on the frosted microslide, and curing at low temperatures, obtain being covered with the slide of ground floor gel; Nutrient solution and 0.8% low melting-point agarose gel preheating and mixing under 36～38 ℃ of temperature that will contain cell to be measured again, be coated in again on the described slide that is covered with the ground floor gel, cover a cover glass, and place under the low temperature and to solidify, remove cover glass then, obtain being covered with the slide of two-layer gel.Described frosted microslide is to add the polishing of water single face through silicon carbide paper (granularity No.380), has the slide of even frosting.Described 1% normal fusing point Ago-Gel (NMP) and 0.8% low melting-point agarose gel (LMPA) all are with the phosphate buffer solution of calcic, magnesium (PBS) is not formulated.Describedly contain the nutrient solution of cell to be measured and the consumption volume ratio of 0.8% low melting-point agarose gel is 1: 3～5.
Described lysis step is that the described slide that is covered with two-layer gel was immersed in 2～6 ℃ the cell pyrolysis liquid 50-70 minute, obtains the slide after the lysis.Described cell pyrolysis liquid be by the Tris damping fluid (10mM, pH=10), volume fraction is that 10% dimethyl sulfoxide (DMSO) and volume fraction are that 1% Triton X-100 forms; Described Tris damping fluid is by 10mM Trizma base, 2.5M NaCl and 100mM Na
2EDTA forms; Described Triton X-100 just joins in the cell pyrolysis liquid before slide immerses.
Described untwisting and electrophoresis step is earlier to wash slide after the described lysis with electrophoresis liquid, then slide placed the electrolytic tank that the precooling electrophoresis liquid is housed to leave standstill 25～40 minutes, carries out electrophoresis again, obtains the slide behind the electrophoresis.Described electrophoresis liquid is by 300mM NaCl and 1mM Na
2The EDTA preparation, pH＞13.Described electrophoretic voltage is 20～30V, and electric current is 250～350mA, and temperature is 2～6 ℃, and electrophoresis time is 25～40 minutes.
In described and washing step, be with the slide behind the described electrophoresis put into the Tris damping fluid (0.4M, pH=7.5) in 4～6 minutes, take out the back and wash repeatedly with distilled water, with in and highly basic and wash dirty Stains off, subsequently it is dried the slide after obtaining neutralizing.
Described fluorescent dye step is to drip the SGD dye solution on the slide after the described neutralization, dyes and uses Tris damping fluid (0.4M, pH=7.5) the unnecessary dye solution of flushing after 8～12 minutes again.
A kind of being specifically designed to implemented the above-mentioned kit that is used for the sample preparation methods of single cell gel electrophoresis graphical analysis, comprises 7 kinds of reagent that configure and the some frosted microslides of handling well: described reagent 1 is asymmetric cyanine class dye solution (SGD dye solution); Described reagent 2 is 1% normal fusing point Ago-Gel; Described reagent 3 is 0.8% low melting-point agarose gel; Described reagent 4 is one of them component of cell pyrolysis liquid, i.e. 10mM Tris damping fluid (10mM Trizma base, 2.5M NaCl, 100mM Na
2EDTA, pH=10); Described reagent 5 is another component of cell pyrolysis liquid, and promptly the 10ml volume fraction is that 10% dimethyl sulfoxide (DMSO) and 1ml volume fraction are 1% TritonX-100, adds before wherein Triton X-100 is to use; Described reagent 6 is electrophoresis liquid, and described electrophoresis liquid is by 300mM NaCl and 1mM Na
2The solution that EDTA mixes; Described reagent 7 is neutralization buffer, and described neutralization buffer is 0.4M, the Tris damping fluid of pH=7.5.
The present invention compared with prior art has following advantage and effect:
(1) the present invention adopts asymmetric cyanine class dye solution (SGD dye solution) to carry out fluorescent dye, because asymmetric cyanine class dye solution is nontoxic, safe and reliable, the experimental waste of generation does not pollute the environment, and has the fluorescence sensitivity higher than EB dyestuff.
(2) gel of shop of the present invention glue system that step is spread only needs two-layer glue, will spread the technology of three layers of gel with the sample slide of traditional single cell gel electrophoresis graphical analysis and compare, and specimen preparation technology of the present invention is simple, easy operating, success ratio height.
(3) sample of technology preparation of the present invention has only two-layer running gel, makes when carrying out the single cell gel electrophoresis graphical analysis, and the decay minimizing to fluorescence has improved the comet brightness of image that generates, and has improved the sensitivity and the picture quality of experiment.
Description of drawings
Fig. 1 adopts the inventive method to variable concentrations H
2O
2The single cell gel electrophoresis image of the sample slide of the human peripheral lymphocyte preparation under the effect.
Fig. 2 adopts the inventive method to variable concentrations H
2O
2The H that the sample slide of the human peripheral lymphocyte preparation under the effect obtains
2O
2Contamination causes lymphocyte DNA damage dose and effect relation figure.
Fig. 3 adopts the inventive method to variable concentrations H
2O
2The single cell gel electrophoresis image of the sample slide of the bone marrow cells in mice preparation under the effect.
Fig. 4 adopts the inventive method to variable concentrations H
2O
2The H that the sample slide of the bone marrow cells in mice preparation under the effect obtains
2O
2Contamination causes bone marrow cell dna damage dosage and effect relation figure.
Embodiment
Below in conjunction with embodiment the present invention is done further detailed description, but embodiments of the present invention are not limited thereto.
Embodiment 1
(1) preparation lymphocyte sample.Do not have smoking history, do not have the male volunteers withdraw venous blood 2ml of the history of taking medicine in the recent period from young healthy, use anticoagulant heparin.Draw dilution blood then and be added in (1: 1) on the lymphocyte separation medium, the centrifugal 20min of 2000r/m.Draw buffy coat, with PBS liquid washed twice, 1500r/m, 10min.Sediment is diluted to suspension with RPMI-1640, makes counting (1 * 107/mL).Then with H
2O
2Contamination: be divided into 6 dosage groups, H
2O
2Final concentration is respectively 50 μ M, 100 μ M, 150 μ M, 200 μ M, 400 μ M, 800 μ M, and other establishes blank group (0 group), places 37 ℃ of water-bath 1h, makes the lymphocyte sample.
(2) shop glue.A frosted microslide 40 ℃ of following preheatings, will evenly be coated on the frosted microslide with 1% normal fusing point Ago-Gel of the not phosphate buffer solution of calcic, magnesium (PBS) preparation then, and solidify at low temperatures, obtain being covered with the slide of ground floor gel.Use 0.8% low melting-point agarose gel (LMPA) of the not phosphate buffer solution of calcic, magnesium (PBS) preparation at 37 ℃ of following preheatings and mixing in 20 μ l lymphocyte samples and 80 μ l, be coated in again on the described slide that is covered with the ground floor gel, cover a cover glass, and place under the low temperature and to solidify, remove cover glass then, obtain being covered with the slide of two-layer gel.
(3) lysis.The slide that will be covered with two-layer gel subsequently immersed in 4 ℃ the cell pyrolysis liquid 1 hour, obtained the slide after the lysis.Described cell pyrolysis liquid be by the Tris damping fluid (10mM, pH=10), volume fraction is that 10% dimethyl sulfoxide (DMSO) and volume fraction are that 1% Triton X-100 forms; Described Tris damping fluid is by 10mM Trizma base, 2.5M NaCl and 100mM Na
2EDTA forms; Described Triton X-100 just joins in the cell pyrolysis liquid before slide immerses.
(4) untwist and electrophoresis.Earlier with the slide after the electrophoresis liquid flushing lysis, then slide is placed the electrolytic tank that the precooling electrophoresis liquid is housed to leave standstill 30 fens kinds, be that 25V, electric current are that 300mA, temperature are under 4 ℃ of conditions at electrophoretic voltage again, carried out electrophoresis 30 minutes, obtain the slide behind the electrophoresis.Described electrophoresis liquid is by 300mM NaCl and 1mM Na
2The EDTA preparation, pH＞13.
(5) neutralization washing.With the slide behind the electrophoresis put into the Tris damping fluid (0.4M, pH=7.5) in 5 minutes, take out the back and wash repeatedly with distilled water, with in and highly basic and wash dirty Stains off, subsequently it is dried the slide after obtaining neutralizing.
(6) fluorescent dye.On the slide after the neutralization, drip the SGD dye solution, dye and use the Tris damping fluid after 10 minutes again (0.4M pH=7.5) rinses out unnecessary dye solution.Described SGD dye solution is the solution that asymmetric cyanine dye SYBR Gold is diluted with 1: 10000 volume ratio with dimethyl sulfoxide (DMSO) (DMSO).
(7) pickup image.Six sample slides that will make at last and a blank slide place that the light with the 495nm wavelength is that exciting light obtains fluoroscopic image and pickup image under the Nikon TE300 inverted fluorescence microscope.
The kit that is used for present embodiment comprises above-mentioned 7 kinds of reagent that configure, and 6 frosted microslides of handling well.Wherein said reagent 1 is the SGD dye solution; Described reagent 2 is 1% normal fusing point Ago-Gel; Described reagent 3 is 0.8% low melting-point agarose gel; Described reagent 4 is one of them component of cell pyrolysis liquid, i.e. 10mM Tris damping fluid (10mM Trizma base, 2.5M NaCl, 100mM Na
2EDTA, pH=10); Described reagent 5 is another component of cell pyrolysis liquid, and promptly the 10ml volume fraction is that 10% dimethyl sulfoxide (DMSO) and 1ml volume fraction are 1% TritonX-100, adds before wherein Triton X-100 is to use; Described reagent 6 is electrophoresis liquid, and described electrophoresis liquid is by 300mM NaCl and 1mM Na
2The solution that EDTA mixes; Described reagent 7 is neutralization buffer, and described neutralization buffer is 0.4M, the Tris damping fluid of pH=7.5.
As shown in Figure 1.The clear picture that the sample of present embodiment preparation shows has higher fluorescent brightness, and with background higher contrast ratio is arranged.The all directed disengaging nucleus of the DNA fragment of damage forms hangover, forms typical comet image.The DNA fragment of each damage shows clear, and higher resolution is arranged.Adopting single cell gel electrophoresis image analysis software (IMI.1 comet image analysis system) to measure its relevant comet parameter can get: comet tail long (Tail Extent), comet tail DNA percentage composition (Tail%DNA), Olive comet tail square (Olive Tail Moment), comet tail head are than (Ratio of Tail/Head).Can calculate the dna damage degree of lymphocyte sample by these comet parameters, as shown in Figure 2, its result shows cell DNA degree of injury and H
2O
2Dosage good dose-effect relationship is arranged.
Embodiment 2
(1) preparation bone marrow cell sample.The femur extracting marrow cell of kunming mice (Guangzhou Experimental Animal Center) from male, body weight 22～25g in 5～6 ages in week with PBS liquid washed twice bone marrow cell, adopted the 1500r/m rotating speed centrifugal 10 minutes then.Sediment after the centrifuging is diluted to suspension with RPMI-1640, makes counting (1 * 107/mL); Then with H
2O
2Contamination also is divided into 6 dosage groups, H
2O
2Final concentration is respectively 50 μ M, 100 μ M, 150 μ M, 200 μ M, 400 μ M, 800 μ M, and other establishes blank group (0 group), places 37 ℃ of water-baths 1 hour, makes the bone marrow cell sample.
(2) shop glue.A frosted microslide 41 ℃ of following preheatings, will evenly be coated on the frosted microslide with 1% normal fusing point Ago-Gel of the not phosphate buffer solution of calcic, magnesium (PBS) preparation then, and solidify at low temperatures, obtain being covered with the slide of ground floor gel.Use 0.8% low melting-point agarose gel (LMPA) of the not phosphate buffer solution of calcic, magnesium (PBS) preparation at 36 ℃ of following preheatings and mixing in 20 μ l lymphocyte samples and 80 μ l, be coated in again on the described slide that is covered with the ground floor gel, cover a cover glass, and place under the low temperature and to solidify, remove cover glass then, obtain being covered with the slide of two-layer gel.
(3) lysis.The slide that will be covered with two-layer gel subsequently immersed in 3 ℃ the cell pyrolysis liquid 70 minutes, obtained the slide after the lysis.Described cell pyrolysis liquid be by the Tris damping fluid (10mM, pH=10), volume fraction is that 10% dimethyl sulfoxide (DMSO) and volume fraction are that 1% Triton X-100 forms; Described Tris damping fluid is by 10mM Trizma base, 2.5M NaCl and 100mMNa
2EDTA forms; Described Triton X-100 just joins in the cell pyrolysis liquid before slide immerses.
(4) untwist and electrophoresis.Earlier with the slide after the electrophoresis liquid flushing lysis, then slide is placed the electrolytic tank that the precooling electrophoresis liquid is housed to leave standstill 40 fens kinds, be that 28V, electric current are that 280mA, temperature are under 3 ℃ of conditions at electrophoretic voltage again, carried out electrophoresis 35 minutes, obtain the slide behind the electrophoresis.Described electrophoresis liquid is by 300mM NaCl and 1mM Na
2The EDTA preparation, pH＞13.
(5) neutralization washing.With the slide behind the electrophoresis put into the Tris damping fluid (0.4M, pH=7.5) in 5 minutes, take out the back and wash repeatedly with distilled water, with in and highly basic and wash dirty Stains off, subsequently it is dried the slide after obtaining neutralizing.
(6) fluorescent dye.On the slide after the neutralization, drip the SGD dye solution, dye and use the Tris damping fluid after 12 minutes again (0.4M pH=7.5) rinses out unnecessary dye solution.Described SGD dye solution is the solution that asymmetric cyanine dye SYBR Gold is diluted with 1: 10000 volume ratio with dimethyl sulfoxide (DMSO) (DMSO).
(7) pickup image.Six sample slides that will make at last and a blank slide place that the light with the 495nm wavelength is that exciting light obtains fluoroscopic image and pickup image under the Nikon TE300 inverted fluorescence microscope.
The kit that is used for present embodiment comprises above-mentioned 7 kinds of reagent that configure, and 6 frosted microslides of handling well.Wherein said reagent 1 is the SGD dye solution; Described reagent 2 is 1% normal fusing point Ago-Gel; Described reagent 3 is 0.8% low melting-point agarose gel; Described reagent 4 is one of them component of cell pyrolysis liquid, i.e. 10mM Tris damping fluid (10mM Trizma base, 2.5M NaCl, 100mM Na
2EDTA, pH=10); Described reagent 5 is another component of cell pyrolysis liquid, and promptly the 10ml volume fraction is that 10% dimethyl sulfoxide (DMSO) and 1ml volume fraction are 1% Triton X-100, adds before wherein Triton X-100 is to use; Described reagent 6 is electrophoresis liquid, and described electrophoresis liquid is by 300mM NaCl and 1mM Na
2The solution that EDTA mixes; Described reagent 7 is neutralization buffer, and described neutralization buffer is 0.4M, the Tris damping fluid of pH=7.5.
As shown in Figure 3.The clear picture of the sample picked-up of present embodiment preparation has higher fluorescent brightness, and with background higher contrast ratio is arranged.The all directed disengaging nucleus of the DNA fragment of damage forms hangover, forms typical comet image.The DNA fragment of each damage shows clear, and higher resolution is arranged.Adopting single cell gel electrophoresis image analysis software (IMI.1 comet image analysis system) to measure its relevant comet parameter can get: comet tail long (Tail Extent), comet tail DNA percentage composition (Tail%DNA), Olive comet tail square (Olive Tail Moment), comet tail head are than (Ratio of Tail/Head).Can calculate the dna damage degree of bone marrow cell sample by these comet parameters, as shown in Figure 4, its result shows cell DNA degree of injury and H
2O
2Dosage good dose-effect relationship is arranged.
Claims (9)
1, a kind of sample preparation methods that is used for the single cell gel electrophoresis graphical analysis, comprise shop glue, lysis, untwist wash with electrophoresis, neutralization, the fluorescent dye step, it is characterized in that: the gel of described shop glue system that step is spread is two-layer, be earlier with the frosted microslide 38～42 ℃ of preheatings, to evenly be coated on the frosted microslide with 1% normal fusing point Ago-Gel of the not phosphate buffer solution preparation of calcic, magnesium then, and solidify at low temperatures, obtain being covered with the slide of ground floor gel; The nutrient solution and 0.8% low melting-point agarose gel preheating and the mixing under 36～38 ℃ of temperature of using the not phosphate buffer solution preparation of calcic, magnesium that will contain cell to be measured again, be coated in again on the described slide that is covered with the ground floor gel, cover a cover glass, and place under the low temperature and to solidify, remove cover glass then, obtain being covered with the slide of two-layer gel; Described fluorescent dye step adopts the SGD dye solution.
2, the sample preparation methods that is used for the single cell gel electrophoresis graphical analysis according to claim 1 is characterized in that: described SGD dye solution is with the solution of asymmetric cyanine dye SYBR Gold with the dimethyl sulfoxide (DMSO) dilution.
3, the sample preparation methods that is used for the single cell gel electrophoresis graphical analysis according to claim 1 is characterized in that: describedly contain the nutrient solution of cell to be measured and the consumption volume ratio of 0.8% low melting-point agarose gel is 1: 3～5.
4, the sample preparation methods that is used for the single cell gel electrophoresis graphical analysis according to claim 1, it is characterized in that: described lysis step, be that the described slide that is covered with two-layer gel was immersed in 2～6 ℃ the cell pyrolysis liquid 50～70 minutes, obtain the slide after the lysis.
5, the sample preparation methods that is used for the single cell gel electrophoresis graphical analysis according to claim 4, it is characterized in that: described cell pyrolysis liquid is the Tris damping fluid by 10mM, pH=10, volume fraction is that 10% dimethyl sulfoxide (DMSO) and volume fraction are that 1% Triton X-100 forms; Described Tris damping fluid is by 10mM Trizma base, 2.5M NaCl and 100mM Na
2EDTA forms; Described Triton X-100 just joins in the cell pyrolysis liquid before slide immerses.
6, the sample preparation methods that is used for the single cell gel electrophoresis graphical analysis according to claim 1, it is characterized in that: described untwisting and electrophoresis step, be earlier to wash slide after the described lysis with electrophoresis liquid, then slide is placed the electrolytic tank that the precooling electrophoresis liquid is housed to leave standstill 25～40 minutes, carry out electrophoresis again, obtain the slide behind the electrophoresis.
7, the sample preparation methods that is used for the single cell gel electrophoresis graphical analysis according to claim 6 is characterized in that: described electrophoresis liquid is by 300mM NaCl and 1mM Na
2The EDTA preparation, pH＞13; The voltage of described electrophoresis is 20～30V, and electric current is 250～350mA, and temperature is 2～6 ℃, and electrophoresis time is 25～40 minutes.
8, the sample preparation methods that is used for the single cell gel electrophoresis graphical analysis according to claim 1, it is characterized in that: in described and washing step, it is the Tris damping fluid 4～6 minutes of the slide behind the described electrophoresis being put into 0.4M, pH=7.5, taking out the back washes repeatedly with distilled water, with in and highly basic and wash dirty Stains off, subsequently it is dried the slide after obtaining neutralizing; Described fluorescent dye step is to drip the SGD dye solution on the slide after the described neutralization, the unnecessary dye solution of Tris damping fluid flushing that dyes and use 0.4M, pH=7.5 after 8～12 minutes again.
9, a kind ofly be used to implement each described kit that is used for the sample preparation methods of single cell gel electrophoresis graphical analysis of claim 1～8, it is characterized in that: comprise 7 kinds of reagent that configure and the some frosted microslides of handling well, described reagent 1 is asymmetric cyanine class dye solution; Described reagent 2 is 1% normal fusing point Ago-Gel; Described reagent 3 is 0.8% low melting-point agarose gel; Described reagent 4 is one of them component of cell pyrolysis liquid, and promptly 10mM is by 10mM Trizma base, 2.5MNaCl and 100mM Na
2The Tris damping fluid of EDTA composition and pH=10; Described reagent 5 is another component of cell pyrolysis liquid, and promptly the 10ml volume fraction is that 10% dimethyl sulfoxide (DMSO) and 1ml volume fraction are 1% Triton X-100, adds before wherein Triton X-100 is to use; Described reagent 6 is electrophoresis liquid, and described electrophoresis liquid is by 300mM NaCl and 1mM Na
2The solution that EDTA mixes; Described reagent 7 is neutralization buffer, and described neutralization buffer is 0.4M, the Tris damping fluid of pH=7.5.
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| CN101907532A (en) * | 2010-03-10 | 2010-12-08 | 刘爱林 | Method for preparing broad bean root tip cell comet assay sample and kit for implementing method |
| CN101865877A (en) * | 2010-06-25 | 2010-10-20 | 西北大学 | Gel paving method by using single cell gel electrophoresis |
| CN103276048A (en) * | 2013-05-07 | 2013-09-04 | 江苏大学 | Preparation method of sample for conveniently and rapidly detecting deoxyribonucleic acid (DNA) cell damage and kit using same |
| CN106868148B (en) * | 2017-03-08 | 2021-02-26 | 中国科学院苏州生物医学工程技术研究所 | Preparation method of cell nucleus with controllable aggregation state and long-term preservation |
| CN110470520A (en) * | 2019-09-16 | 2019-11-19 | 武汉赛维尔生物科技有限公司 | Liquid method is changed in a kind of comet |
| CN113405881A (en) * | 2021-06-10 | 2021-09-17 | 华东理工大学 | Rapid detection method and kit for single cell DNA damage |
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