CN106018529A - Kit using single cell gel electrophoresis for detecting cell DNA damage and method - Google Patents
Kit using single cell gel electrophoresis for detecting cell DNA damage and method Download PDFInfo
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- CN106018529A CN106018529A CN201610531656.5A CN201610531656A CN106018529A CN 106018529 A CN106018529 A CN 106018529A CN 201610531656 A CN201610531656 A CN 201610531656A CN 106018529 A CN106018529 A CN 106018529A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
- G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44756—Apparatus specially adapted therefor
- G01N27/44791—Microapparatus
Abstract
The invention provides a kit using single cell gel electrophoresis for detecting cell DNA damage and a method. The method includes gel laying, splitting, unwinding, electrophoresis, neutralizing and washing, drying and dyeing, a fluorescence microscope is used for observing a dyeing result and taking photos, and the damage degree of cell DNA is analyzed and evaluated. As three layers of gel need to be laid on a glass slide during sample preparation, a traditional single cell gel electrophoresis technology has the disadvantages of being complex in operation and prone to degumming, and sample images are unclear; while only one layer of gel needs to be laid on a glass slide, the kit has the advantages of being simple in sample preparing process, low in degumming rate, easy to operate, low in experiment cost and the like.
Description
Technical field
The present invention relates to biological technical field, particularly relate to the test kit of a kind of single cell gel electrophoresis detection DNA Damage
And method.
Background technology
Along with the accelerated development of China's economic, environmental pollution is also on the rise.At present, the environmental pollution in global range is to human body
Health also receives more and more attention.Wherein, environmental contaminants cause the damage of DNA can affect the hereditary stability of cell,
And offspring may be produced impact, cause anemia of pregnant woman to miscarry, stillborn fetus, monster and some hereditary etc..DNA due to cell
Can damage can cause disease, threatens human health, therefore detects DNA Damage, and understand this damage and draw
The change playing cell event just seems particularly significant.
1976, COOK et al. invented a kind of method studying DNA Damage, i.e. single cell gel electrophoresis technique
(Cook P.R,Brazell I.A,Jost E.Journal of Cell Science,1976,22(2):303-324.).1978, Rydberg
Report for the first time on the basis of above-mentioned experiment with Johanson and directly in the level of individual cells, damage is carried out detection by quantitative
(Rydberg B,Johanson K J.DNA Repair Mechanisms,1978(03):465-468.).Based on the above method,
Ostling and Johanson established microgel electrophoretic techniques (Ostling O, Johanson K J.Biochemical and in 1984
Biophysical Research Communications, 1984,123 (1): 291-298.), this technology adds electrophoresis step in solution supination
Suddenly, make part move from the nucleus Ghandler motion that faces south, be similar to the tail of comet to be formed, but the cracking under neutrallty condition and electrophoresis
The double-stranded DNA part in cell can only be detected, it is impossible to measure single-strand break.Therefore Singh in 1988 establishes alkalescence comet
Experiment (pH > 13.0), this experiment can detect single, double chain break, and (some damage factor can result in alkalescence point of instability
Depurination or the generation in de-pyrimidine site, form alkalescence point of instability, and it can be converted into DNA under basic conditions
Fracture) so that the sensitivity of detection is greatly improved (Singh N P, McCoy M T, Tice R R, et al.Experimental
Cell Research,1988,175(1):184-191.).Since alkalescence comet comes out, each laboratory is by this skill
Art is widely used in detecting the damage of cell DNA, meanwhile, is also constantly improveing the method, is enabling
Detect intracellular different DNA damage, such as single double chain break, the oxidative damage of base, DNA-DNA/DNA-albumen
Matter-medicine crosslinking and reparation etc., but, the method there is also series of malpractice, such as, operate complexity, easy degumming, sample
Not fogging clear etc., thus affect the interpretation of result of image.
Summary of the invention
The shortcoming of prior art in view of the above, it is an object of the invention to provide a kind of single cell gel electrophoresis detection cell DNA
The test kit of damage and method, be used for solving in prior art operating that complexity, the easy degumming of cell, sample image are unintelligible etc. asks
Topic.
For achieving the above object and other relevant purposes, first aspect present invention provides a kind of single cell gel electrophoresis detection cell DNA
The test kit of damage, including following reagent: low melting-point agarose gel, cell pyrolysis liquid, alkaline electrophoresis buffer, dyeing liquor.
Low melting-point agarose refers to that gelation temperature is 26 DEG C-30 DEG C (1.5%gel), the agarose of fusion temperature≤65 DEG C (1.5%gel).
Further, low melting-point agarose gel refers to low melting-point agarose and without calcium, the mixed liquor of the PBS of magnesium, low melting point fine jade
In sepharose, the mass concentration of low melting-point agarose is 0.75%；Cell pyrolysis liquid includes 2.5mmol/L NaCl, 100mmol/L
Na2-EDTA, 100mmol/L Tris, pH is 10, and adding Triton X-100 before use to volume fraction is 1%, adds simultaneously
DMSO is 10% to volume fraction；Alkaline electrophoresis buffer includes 200mmol/L NaOH, 1mmol/L EDTA；Dyeing
Liquid is nucleic acid gel electrophoretic pigment Gelgreen or Genelight.
Second aspect present invention provides the method for a kind of single cell gel electrophoresis detection DNA Damage, comprises the steps:
1) paving glue: on microscope slide lay coverslip, and formed with microscope slide as bottom surface, the coverslip sulculus as sidewall, the most micro-
Electrophoresis tank；DNA sample suspension and low melting-point agarose gel are mixed to obtain cell mixture, uses described cell mixture to exist
, there is gap in electrophoresis tank middle berth glue, the cell mixture on microelectrophoresis bath penetrates into both sides before solidification between coverslip and microscope slide
Gap in, there is gap between coverslip and microscope slide, the cell mixture on microelectrophoresis bath penetrates between both sides before solidification
In gap；Cover-glass thickness is less, it is simple to cut, and the slide being bonded on microscope slide can be complete coverslip, it is also possible to be to cut out
It is switched to the arrow gauge of certain size.On microelectrophoresis bath, the both sides of gel are fixed, the problem efficiently solving easy degumming.
2) cracking: the microscope slide after paving glue is immersed in cell pyrolysis liquid and cracks.
3) untwist, electrophoresis, neutralization washing: the microscope slide after cracking is immersed in alkaline electrophoresis buffer and untwists, after untwisting
Electrophoresis, after electrophoresis terminates, neutralizes washed；Microscope slide therein is preferably smooth microscope slide, it is simple to the collection of image and
Analyze, improve the sensitivity of comet detection.
4) it is dried: the microscope slide after neutralizing washing is dried.
5) dyeing: the dyeing of agar circle, dried, the degree of injury of assay cell DNA.
Further, step 1) in, DNA Damage sample suspension refer to be suspended with DNA Damage sample and without calcium,
The PBS of magnesium, low melting-point agarose gel refers to low melting-point agarose and without calcium, the mixed liquor of the PBS of magnesium, low melting-point agarose
In gel, the mass concentration of low melting-point agarose is 0.75%, DNA Damage sample suspension and the body of low melting-point agarose gel
Long-pending ratio is 1:10.
Further, in DNA Damage sample suspension, cell density is 1 x 105cells/ml。
Further, the temperature of the PBS being suspended with DNA Damage sample is 37 DEG C, prevents cell heat shock.
Preferably, step 1) in, the manufacture method of microelectrophoresis bath is as follows: coverslip is divided into fillet, is seamless bonding by fillet
At the middle part of microscope slide, newer coverslip is bonded at the two ends of microscope slide, leaves spacing between coverslip and fillet, form micro-electricity
Swimming groove.It is of course also possible to do not use fillet, microscope slide is cut out short, at the two ends adhesive cover slide of microscope slide, two coverslipes
Between leave spacing, form microelectrophoresis bath, between coverslip and microscope slide, leave gap, it is simple to low melting-point agarose gel penetrates into
Wherein, the sample in microelectrophoresis bath is played fixation.
Preferably, described fillet is rectangle fillet.
Preferably, the width of microelectrophoresis bath is 10mm.
Preferably, step 1) in, the glue used time bonding is UFO super glue, AB glue or neutral density glass glue.
Further, step 5) in, during the dyeing of agar circle, the dyeing liquor chosen be nucleic acid gel electrophoretic pigment Gelgreen or
Genelight。
Further, step 1) in, cell mixture is laid in microelectrophoresis bath, cell mixture penetrates into two before solidification
In the coverslip of end, 4 DEG C of lucifuge solidification 25-30min.
Further, step 2) in, described in be cracked into lucifuge cracking, cell pyrolysis liquid includes 2.5mmol/L NaCl, 100mmol/L
Na2-EDTA, 100mmol/L Tris, pH is 10；Adding Triton X-100 before use to volume fraction is 1%, adds simultaneously
DMSO is 10% to volume fraction；Lytic conditions is: temperature 4 DEG C, time 1-2h.
Further, step 3) in, the cell after cracking is cleaned with distilled water, then is placed in alkaline electrophoresis buffer
Lucifuge is untwisted, and the alkaline electrophoresis buffer used when untwisting includes 200mmol/L NaOH, 1mmol/L EDTA, cell solution
Rotation condition is: temperature 4 DEG C, time 20-40min.
Further, step 3) in, solve supination, microscope slide is placed in electrophoresis tank, adds new pre-cooling alkalescence running buffer
Liquid carries out electrophoresis, and deposition condition is: temperature 4 DEG C, voltage 21V, time 20-30min；New alkaline electrophoresis buffer includes
200mmol/L NaOH, 1mmol/L EDTA, the buffer i.e. used during electrophoresis is identical with the buffer used when untwisting.
Tris-HCl buffer is used when neutralizing washing；Step 4) in, the baking temperature of microscope slide is 37-45 DEG C, and drying time is
10-15min。
As it has been described above, a kind of single cell gel electrophoresis detection test kit of DNA Damage of the present invention and method, have with
Lower beneficial effect: (1) implementation process is simple and easy to control；(2) the method effectively reduces degumming rate, and sample image becomes apparent from；This
Bright effective experiment risk that reduces, nucleic acid electrophoresis dyestuff Gelgreen or Genelight of dyeing liquor safety replaces ethidium bromide dye,
Color is more preferable, decreases the body harm to experimenter.
Traditional single cell gel electrophoresis technique when prepared by sample need on microscope slide paved three layers of gel, have operation complicated,
Shortcomings such as easily degumming, sample image are unintelligible, and the present invention uses homemade microscope slide, only need on microscope slide paved one layer
Glue, has the advantages such as sample preparation technology is simple, degumming rate is low, easily operated, experimental cost is low, image is clear.
Accompanying drawing explanation
Fig. 1 is shown as self-control microscope slide used during cell embedding.
Fig. 2 is shown as the comet image photograph of matched group.
Fig. 3 is shown as the comet image photograph after 5mg/L graphene oxide exposes 293T cell 24h.
Fig. 4 is shown as the comet image photograph after 25mg/L graphene oxide exposes 293T cell 24h.
Fig. 5 is shown as the comet image photograph after 50mg/L graphene oxide exposes 293T cell 24h.
Fig. 6 is shown as the comet image photograph after 100mg/L graphene oxide exposes 293T cell 24h.
Detailed description of the invention
Below by way of specific instantiation, embodiments of the present invention being described, those skilled in the art can be by disclosed by this specification
Content understand other advantages and effect of the present invention easily.The present invention can also be added by the most different detailed description of the invention
To implement or application, the every details in this specification can also be based on different viewpoints and application, in the essence without departing from the present invention
Various modification or change is carried out under god.
HEKC is selected to cultivate, at 37 DEG C, 5%CO2, saturated humidity CO2 gas incubator in cultivate, treat thin
When born of the same parents' length is to logarithmic (log) phase, cell is inoculated in six orifice plates, adds (the preparation of culture fluid: according to required cultivation of 3mL culture fluid
The volume of liquid is separately added into Gibco-1640 culture medium, hyclone and Penicillin Streptomycin Solution (100X), makes each composition
The volume fraction of composition reaches 49%, 10% and 1%).
Expose the preparation of liquid: weigh 0.005g graphene oxide and be placed in brown bottle, add 1ml distilled water, ultrasonic after shaking up
30min, makes graphene oxide be dispersed in water.Pipette the most respectively 60 μ l, 30 μ l mother solution in 4mlEP pipe, add
Corresponding culture fluid, is configured to the exposure liquid that 3ml concentration is 100mg/l, 50mg/l, then is diluted to by the exposure liquid of 50mg/l
The exposure liquid of 25mg/l and 5mg/l.
After the degree of converging of cell length to about 70%, cell culture fluid is discarded, add the exposure liquid of respective concentration, expose 24h.
After cell exposes, exposure liquid is discarded, with 1 × PBS cell two to three times, then with 1 × PBS of pre-cooling by cell
Piping and druming is got off, by resuspended for cell to 1 x 105Cells/ml, whole process wants lucifuge.
The making of microscope slide: with diamant, coverslip is divided into the rectangle fillet of two 20mm × 10mm, then fillet is used
UFO glue adheres to the middle part of clean smooth microscope slide, then with UFO glue, complete coverslip is adhered to microscope slide two ends,
Make between coverslip and fillet, to leave a part of space, i.e. microelectrophoresis bath (in the present embodiment, the width of electrophoresis tank is 10mm,
The length of electrophoresis tank is consistent with the width of microscope slide).Middle part rectangle coverslip fillet and microscope slide between be close to completely, i.e. without
Stitch bonding, play buffer action, form two microelectrophoresis baths, obtain two groups of samples, between coverslip and the microscope slide at two ends all
There is certain gap so that cell mixture can penetrate into wherein before solidification, the microscope slide prepared is as shown in Figure 1.Certainly,
Other glue such as AB glue, neutral density glass glue can also be used during adhesive cover slide, but, AB glue to be allocated when using,
Inconvenient operation；Neutral density glass is gluing connect after, coverslip is difficult to take off, be not easy to take pictures end after to microscope slide and lid
The inner side of slide is thoroughly cleaned, and therefore, the present embodiment preferably employs UFO super glue (model: Art.NO.01003),
UFO super glue rapid solidification in atmosphere, easy to operate when adhesive cover slide, after experiment terminates, use alcohol-pickled load
Slide about 3-5min, then rinse with clear water, coverslip can be taken off from microscope slide, simple to operate.The thickness of coverslip
Relatively thin, generally at 0.13-0.20mm, it is simple to cut.The slide of electrophoresis tank both sides plays the effect of fixing agarose, effectively prevents
Degumming, the shape of electrophoresis tank is not limited to the rectangle shown in Fig. 1, and other are any can reach the electrophoresis tank shape of above-mentioned purpose also
Within protection scope of the present invention.
The preparation of cell pyrolysis liquid: cell pyrolysis liquid can first prepare stock solution, prepares application liquid before using.Cell pyrolysis liquid is stocked
The preparation of liquid: stock solution includes 2.5mM NaCl, 100mM Na2-EDTA, 100mM Tris, arrive with NaOH regulation pH
10, high temperature sterilize, room temperature preservation.The preparation of cell pyrolysis liquid application liquid: add Triton X 100 before use in stock solution
Being 1% to volume fraction, being simultaneously introduced DMSO (dimethyl sulfoxide) is 10% to volume fraction, is placed in 4 DEG C of Refrigerator stores.
The system of the preparation of alkaline electrophoresis buffer: 1L is 8gNaOH+2mlEDTA (500mM, pH=8), molten at NaOH
DH is added after solution2O to 1L, solution is now with the current, is cooled to 4 DEG C.
The preparation of low melting-point agarose gel: preparation mass concentration is the low melting-point agarose solution of 0.75%, specifically, weighs
Required low melting point agar powder, puts in EP pipe, adds 1 × PBS, stirring while adding, makes agarose be thoroughly dispersed in PBS
In solution, with boiling water bath, it being heated about 7min, limit heating edge is shaken, and treats that agarose is completely dissolved, then puts it into water-bath
Its temperature is made to reduce to 37 DEG C in Guo, the low melting-point agarose gel preventing cell heat shock, prepared mass concentration from being 0.75%.
Paving glue: 1 × PBS will be suspended in (without Ca2+, Mg2+) cell and low melting-point agarose gel mix with the volume ratio of 1:10
Even, prepare cell mixture, take 100 μ l cell mixtures and be laid in microelectrophoresis bath so that cell mixture oozes before solidification
Enter in the coverslip at two ends, 4 DEG C of lucifuge solidification 30min, with gauze, microscope slide is encased after it solidifies, prevent degumming.
Cracking: the microscope slide of embedded cell is completely immersed in cell pyrolysis liquid, 4 DEG C of lucifuge cracking 90min.
Untwist: the distilled water of cell pre-cooling after cracking is cleaned two to three times, then is placed on the alkaline electrophoresis buffer of pre-cooling
In 4 DEG C of unwindase 12 0-30min.
Electrophoresis: lain in by microscope slide in electrophoresis tank, the concentration adding new preparation is 200mmol/L NaOH, 1mmol/L EDTA
Alkaline electrophoresis buffer, 4 DEG C of 21V electrophoresis 30min.
Neutralize: clean slide three times by Tris-HCl neutralization buffer, each 5min.Tris-HCl neutralization buffer is 0.4mol/L
Tris-HCl, regulates pH to 7.5,4 DEG C of preservations with HCl.
It is dried: slide is immersed dH2In O twice, each 5min, it is then immersed in 70% (volume fraction) of 4 DEG C of pre-coolings
5min in ethanol solution.At 37-45 DEG C, toast 10-15min, slide is dried.The method can make the single distribution of cell,
It is easy to observe.Sample after drying can drying at room temperature preserve before observation.
Dyeing: taking 100 μ L Genelight nucleic acid electrophoresis dyeing liquors and be coated on the dry agar circle of circle, lucifuge places 4 DEG C of refrigerators
Dyeing 20min, more unnecessary dyeing liquor is removed gently with suction nozzle, the placement of room temperature lucifuge is completely dried to slide.
Take pictures: with fluorescence microscope coloration result and shoot photo, as shown in Fig. 2-Fig. 6.
Data analysis: carry out graphical analysis with CASP software, it is thus achieved that each relevant parameter of comet, major parameter has afterbody DNA
(DNAT, tail DNA), Olive tail square (OTM, Olive tail moment), tail length (TL, Tail Length), tail
Square (TM, Tail moment), by the statistical analysis to these parameters, obtains under variable concentrations, the DNA of 293T cell
Degree of injury.Be as shown in table 1 variable concentrations graphene oxide to 293T cell afterbody DNA content, DNA migration length,
Tail square and the impact of Olive tail squareData show, the DNA damage of 293T cell and the exposure of graphene oxide
Good dose-effect relationship is presented between concentration.
Table 1
***P<0.001
In sum, the present invention uses homemade microscope slide, only need on microscope slide paved one layer of glue, to paving glue, crack, solve
The steps such as rotation, electrophoresis are optimized so that single cell gel electrophoresis technique is simple to operate, image is clear, use the nucleic acid of safety
Electrophoretic pigment Gelgreen or Genelight replaces ethidium bromide dye, and Color is more preferable, decreases the health to experimenter
Harm.
The principle of above-described embodiment only illustrative present invention and effect thereof, not for limiting the present invention.Any it is familiar with this skill
Above-described embodiment all can be modified under the spirit and the scope of the present invention or change by the personage of art.Therefore, such as
All that in art, tool usually intellectual is completed under without departing from disclosed spirit and technological thought etc.
Effect is modified or changes, and must be contained by the claim of the present invention.
Claims (10)
1. the test kit of single cell gel electrophoresis detection DNA Damage, it is characterised in that include following reagent: low melting point fine jade
Sepharose, cell pyrolysis liquid, alkaline electrophoresis buffer, dyeing liquor.
2. the method for a single cell gel electrophoresis detection DNA Damage, it is characterised in that comprise the steps:
1) paving glue: on microscope slide lay coverslip, and formed with microscope slide as bottom surface, the coverslip sulculus as sidewall, the most micro-
Electrophoresis tank；DNA sample suspension and low melting-point agarose gel are mixed to obtain cell mixture, uses described cell mixture to exist
, there is gap in electrophoresis tank middle berth glue, the cell mixture on microelectrophoresis bath penetrates into both sides before solidification between coverslip and microscope slide
Gap in；
2) cracking: the microscope slide after paving glue is immersed in cell pyrolysis liquid and cracks；
3) untwist, electrophoresis, neutralization washing: the microscope slide after cracking is immersed in alkaline electrophoresis buffer and untwists, after untwisting
Electrophoresis, after electrophoresis terminates, neutralizes washed；
4) it is dried: the microscope slide after neutralizing washing is dried；
5) dyeing: the dyeing of agar circle, dried, the degree of injury of assay cell DNA.
The method of single cell gel electrophoresis the most according to claim 2 detection DNA Damage, it is characterised in that: step 1)
In, DNA Damage sample suspension refers to be suspended with DNA Damage sample and without calcium, the PBS of magnesium, low melting point
Agarose gel refers to low melting-point agarose and without calcium, the mixed liquor of the PBS of magnesium, low melting point in low melting-point agarose gel
The mass concentration of agarose is 0.75%, and DNA Damage sample suspension is 1 with the volume ratio of low melting-point agarose gel:
10。
The method of single cell gel electrophoresis the most according to claim 2 detection DNA Damage, it is characterised in that: step 1)
In, the manufacture method of microelectrophoresis bath is as follows: coverslip is divided into fillet, and fillet is seamless bonding the middle part at microscope slide, then
New coverslip is bonded at the two ends of microscope slide, between coverslip and fillet, leaves spacing, form microelectrophoresis bath.
The method of single cell gel electrophoresis the most according to claim 4 detection DNA Damage, it is characterised in that: step 1)
In, the glue used time bonding is UFO super glue, AB glue or neutral density glass glue.
The method of single cell gel electrophoresis the most according to claim 2 detection DNA Damage, it is characterised in that: step 5)
In, during the dyeing of agar circle, the dyeing liquor chosen is nucleic acid gel electrophoretic pigment Gelgreen or Genelight.
The method of single cell gel electrophoresis the most according to claim 2 detection DNA Damage, it is characterised in that: step 1)
In, cell mixture is laid in microelectrophoresis bath, cell mixture penetrates in the coverslip at two ends before solidification, keeps away for 4 DEG C
Photocoagulation 25-30min.
The method of single cell gel electrophoresis the most according to claim 2 detection DNA Damage, it is characterised in that: step 2)
In, described in be cracked into lucifuge cracking, cell pyrolysis liquid includes 2.5mmol/L NaCl, 100mmol/L Na2-EDTA、
100mmol/L Tris, pH are 10；Adding Triton X-100 before use to volume fraction is 1%, adds DMSO to body simultaneously
Fraction is 10%；Lytic conditions is: temperature 4 DEG C, time 1-2h.
The method of single cell gel electrophoresis the most according to claim 2 detection DNA Damage, it is characterised in that: step 3)
In, the cell after cracking is cleaned with distilled water, then is placed on lucifuge in alkaline electrophoresis buffer and untwists, use when untwisting
Alkaline electrophoresis buffer include that 200mmol/L NaOH, 1mmol/L EDTA, the cell condition of untwisting are: temperature 4 DEG C,
Time 20-40min.
The method of single cell gel electrophoresis the most according to claim 2 detection DNA Damage, it is characterised in that: step
Rapid 3), in, solve supination, microscope slide is placed in electrophoresis tank, adds new pre-cooling alkaline electrophoresis buffer and carry out electrophoresis, electricity
Swimming condition is: temperature 4 DEG C, voltage 21V, time 20-30min；Tris-HCl buffer is used when neutralizing washing；Step
Rapid 4) in, the baking temperature of microscope slide is 37-45 DEG C, and drying time is 10-15min.
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| CN112798594A (en) * | 2020-12-29 | 2021-05-14 | 浙江海洋大学 | Microscopic measurement method for branch and corner type shell and flap distance |
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| CN112798594B (en) * | 2020-12-29 | 2023-04-14 | 浙江海洋大学 | Microscopic measurement method for branch and corner type shell and flap distance |
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