CN101865877A - Gel paving method by using single cell gel electrophoresis - Google Patents
Gel paving method by using single cell gel electrophoresis Download PDFInfo
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- CN101865877A CN101865877A CN201010209930A CN201010209930A CN101865877A CN 101865877 A CN101865877 A CN 101865877A CN 201010209930 A CN201010209930 A CN 201010209930A CN 201010209930 A CN201010209930 A CN 201010209930A CN 101865877 A CN101865877 A CN 101865877A
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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
The present invention relates to a kind of gel paving method by using single cell gel electrophoresis, belong to biomolecule cell experiment technical field.
Background technology
The degree of dna damage determines cell life and death to a certain extent with repairing possibility, and its detection method is one of problem of paying close attention to of people always.Though it is multiple that the technology of this respect has, easy, quick, economic and responsive method should be first elected single cell gel electrophoresis (SCGE), or claims comet (comet) electrophoresis.SCGE is first technology that can effectively detect the individual cells dna damage, has been applied to dna damage and reparation research that various karyocytes are induced through the physics and chemistry factor at present.
Single cell gel electrophoresis is that people such as Singh introduce alkaline environment on this basis and grow up by Ostling and johanson invention.The DNA chain break that causes after the various physical and chemical factor function cells can detect with this method, and on statistical basis degree of injury is made assessment.Its ultimate principle is that the DNA supercoil is rely the nuclear skeleton that adheres to or paralinin after high salt and detergent cracking with it, in alkalescence (pH＞12.3) environment, untwist and electrophoresis again, the DNA fragment of fracture will be released and the anode swimming, and microscopically is observed and is " comet " shape after the fluorescent dye.
Steps such as the experimental implementation of the various comets experiments of adopting at present comprises mainly that acquisition, the preparation of electrophoresis offset plate, lysis, the DNA sex change of single-cell suspension liquid are untwisted, electrophoresis, neutralization, dyeing and observation.
Film preparation promptly is that the cell suspending liquid that will obtain mixes with low melting-point agarose and is laid on microslide, and this is a critical step in the SCGE technology.Plate-making method has 3 kinds: " sandwich " gel, double-deck gel and individual layer gel.The total principle of making sheet process is: obtain the firm stable gel, avoid damage and the reparation of extra DNA.
1, " sandwich " gel: " sandwich " formula structure of 3 layers of agarose is present the most frequently used experimental technique, in this " sandwich " formula structure, the 1st layer is agarose (NMA) layer of normal fusing point, and its effect is to make the layers 2 and 3 agarose be attached on the microslide securely; The 2nd layer of agarose is to comprise nuclear low melting-point agarose (LMA); The 3rd layer is the LMA layer, can prevent that the 2nd layer of cell DNA in the agarose breaks away from gel when alkali treatment and electrophoresis, and the 2nd layer of cell in the agarose all is under the identical conditions.
In order not make the glue accidental, this method a lot of improvement have also been carried out.When ground floor glue is made in the shop, Navarrete (Mutat.Res, 389:271～277,1997.) etc. adopted following method: microslide vertically inserts among 65 ℃ the 0.65%NMA, take out the vertical placement in back, it is solidified, can obtain the agarose layer of the strong uniformity of adhesive force.Purple strong grade the in the Meng (China's chemistry and molecular biology journal, 14 (5): 604-609,1998.) adopts the NMA with preheating to drop on the microslide, and 4 ℃ solidify it, and the microslide preheating can increase the adhesion between agarose and the microslide.In order to increase the adhesion between low melting-point agarose and nucleus suspending liquid and microslide, adopt the microslide glue plate of complete wear sand in addition more.Liu Feng people such as (Nanjing Medical University's journal, 19 (4): 326-327,1999) is for can be at two samples in a microslide upper berth, after the ground floor gelling is solid, draw the wide glue of 2mm down in microslide central authorities with sharp cutter, gel is divided into 2, be used to annotate the target cell of 2 samples.And adopt the method do not add cover glass, when getting cover glass, glue is torn to shreds avoiding.
2, double-deck gel: on " sandwich " gel basis, through repeatedly practice, find only to spread first and second layers of gel, test findings is not impacted, this method has been simplified shop glue program.
3, individual layer gel:, can on slide, make a groove and carry out the individual layer encapsulating for improving the shortcoming that glue-line easily separates with slide.The microelectrophoresis bath of Liu Qiang people such as (Chinese radiation hygiene, 15 (2): 153-155,2006) invention need be transformed earlier microslide, and this method glue is rapid, simple to operate, and degumming rate is almost nil.
In order to reduce degumming rate, some researchist makes the frosted microslide by oneself, and (granularity, NO.380) microslide that ordinary optical microscope is used (thinner thickness is advisable) single face adds water and gently grinds, and makes the milled sand surface of formation evenly fine and closely woven with waterproof abrasive paper.This abrasive segments is used for sandwich glue, double-deck glue or the preparation of individual layer glue, can effectively reduce degumming rate, but need the fresh sheet of readding, can not in time read sheet, need ready-made film is put into the 4 ℃ of preservations of magazine (PBS infiltration) that are covered with wet gauze if promptly finish the back at electrophoresis.Keep gauze humidity, film can be placed a week.
Because wet gel sheet difficulty aspect storage and transport is bigger, and the storage time is long more, the possibility of DNA diffusion is just big more, and the result is unstable more.Existing both at home and abroad research uses methanol dehydration seasoning and ethanol dehydration seasoning to preserve gel film.Film dehydrates method and is keeping having brought convenience to the researchist on the reliable and stable basis of test findings.
" sandwich " gel plate-making method complicated operation, length consuming time is come unstuck easily, the limited amount of preparation sample on the microslide.Double-deck gel is improved on the basis of three layers of gel basically, still receives the quantitative limitation of preparation sample number.
Summary of the invention
The purpose of this invention is to provide a kind of simply, gel paving method by using single cell gel electrophoresis fast, untwist, link such as cracking, electrophoresis and neutralization is difficult for coming unstuck, and dehydrates easily, conveniently read sheet, it is loaded down with trivial details to overcome existing preparing gel technical process, length consuming time, the degumming rate height is difficult for dry deficiency.
Implementation procedure of the present invention is as follows:
A kind of gel paving method by using single cell gel electrophoresis comprises the steps:
(1) be in 0.2%～1.0% the normal fusing point agarose solution after the submergence with the microslide of cleaning at mass percent concentration, 37 ℃ of dry for standby;
(2) with cell suspension and mass percent concentration be 1% low melting-point agarose solution with 1: 1 mixing of volume ratio, 10～40 μ l mixed liquors are dropped on the microslide behind the above-mentioned plated film;
(3) the shop film, at the uniform velocity will be spread film and drag backward near cell suspension with 30～miter angle, and suspension is paved into film like along shop film front end expansion until cell suspension, and shop glue is finished, and 4 ℃ solidify 2～3min.
Described shop film is that quality is pliable and tough, waterproof, the avirulence material, and as the PVC material, its thickness is 0.15～0.35mm.
Described shop film is trapezoidal, wide 1cm and the 0.7cm of being respectively in two ends, and height is 4cm, is used for the vary in size shop system of glue of area; Described shop film can also be strip, and long is 4cm, and wide is 0.7～1cm.
Advantage of the present invention and good effect: 1, gel paving method by using single cell gel electrophoresis of the present invention does not come unstuck.Success ratio is up to 99.99%; 2, shop glue speed is fast, and it is particularly convenient to handle batch samples; 3, increased contained sample number on every microslide, a conventional microslide (25mm x 75mm) can be spread 5～7 samples, and each sample glued membrane area is about 10mm x 20mm; 4, dry plate and wet sheet microscopy all can.Because glue-line is thin, very easily dehydrate, especially be fit to the dry plate microscopy.The gel of doing makes all comets all at same surface level, and the refocus that need not stop is not given a mark easily than fresh gel, brings very big facility to testing crew; 5, method is simple, is convenient to grasp, and need not buy or make by oneself the frosted microslide, perhaps conventional light face microslide is carried out the outward appearance transformation, and is with low cost.
Description of drawings
The side schematic view of microslide when Fig. 1 spreads glue for the present invention;
Fig. 2 is a microslide synoptic diagram of completing sample;
Fig. 3 is the comet electrophoretogram (400x) of woods tobacco protoplast after with uv b radiation: A is 0s, and B is 5s, and C is 10s, and D is 30s.
Specific embodiments
As shown in Figure 1, the shop film, at the uniform velocity will be spread film and drag backward near cell suspension with 30～miter angle, and suspension is paved into film like along shop film front end expansion until cell suspension, and shop glue is finished, and 4 ℃ solidify 2～3min.Use shop of the present invention gluing method, can be at a conventional microslide (25mm x 75mm) 5～7 samples in upper berth (Fig. 2).
Be that material carries out single cell gel electrophoresis and specifies the present invention with the woods tobacco protoplast below.1, tobacco protoplast is free
For the examination material is the aseptic seedling of woods tobacco (Nicotiana sylvestris).Get fully extended leaf, cut off blade, stay vein, tear the vein epidermis off, tiltedly cut into chunks again, add enzyme liquid (2% cellulase Onozuka R-10,0.5% hemicellulase, 0.5% pectase Y-23,0.5% macerozyme R-10, the CaCl of 0.16mol/L
2, 0.1%MES, 0.4mol/L sweet mellow wine, pH 5.8-6.2) and enzymolysis 6-12h in 28 ℃ of dark.Bioplast suspending liquid behind the enzymolysis is filtered the centrifugal 5min of 600r/min with 200 order stainless steel sifts.The bioplast of precipitation is suspended in the CaCl of 2mL 0.16mol/L
22H
2Among the O (pH 5.8-6.2), slowly inject 20% sucrose solution 6mL, the centrifugal 5min of 600r/min to the centrifuge tube bottom with syringe (loading onto the minute hand head).To occur the pure bioplast band of one deck between the interface of two phase liquid, suction pipe is collected and is used 8mL 0.16mol/L CaCl again
22H
2O (pH 5.8-6.2) suspends standby.
2, main agents and instrument
Low melting-point agarose (Amresco packing); Normal fusing point agarose (Spanish is original-pack); TritonX-100 (Roth product); Dimethyl sulfoxide (DMSO) (Amresco is original-pack), Sodium Inosinate (Sigma packing); It is pure that all the other biochemical reagents are analysis.Electrophoresis apparatus (Amersham Biosciences) electrophoresis tank: Tanon HE120; Fluorescent microscope: Leica DMLB 2 (Leica company); The A650 of Canon digital camera, comet image analysis software: CASP software (casp-1.2.2, http://casp.sourceforge.net/index.php downloads).
3, comet electrophoresis
1. coating method prepares primer, and the microslide of cleaning behind the submergence 1min, is put 37 ℃ of incubator bakings and spent the night in 0.6% normal fusing point agarose; During experiment with cell suspension and 1% low melting-point agarose (37 ℃) with 1: 1 ratio mixing in centrifuge tube, with the rifle head that cuts off the tip mixed liquor is dropped on the microslide behind the plated film, shop film (wide 1cm and the 0.7cm of being respectively, height is the thick PVC sheet of 0.25mm of 4cm) with the close cell suspension in 40 degree angles, drop-down gently with the shop film, again microslide is rocked gently, glue is tiled on the microslide.4 ℃ leave standstill 3min and make and contain the cell glue-line.2. cracking, (2.5molL
-1NaCl, 100mmolL
-1Na
2EDTA, 10mmolL
-1Tris, pH10,1% sodium sarcosinate are with before adding 1%TritonX-100,10%DMSO) 4 ℃, 1h.3. washing, 4 ℃ of washings of tri-distilled water 3 times, 5min/ time.4. untwist (1mMNa in the microslide discharge swimming damping fluid
2EDTA, 300mMNaOH, pH 〉=13) 4 ℃, 15min.5. electrophoresis, 4 ℃, 15min, 0.74Vcm
-1(26V, 300mA).6. neutralization, 400mMTris damping fluid, pH7.5,3 times, 5min/ time.7. ice-cold absolute ethyl alcohol dehydration 10min, room temperature is dried.8. dyeing (EB, 20 μ g/ml, 20 μ l), microscopy is got 50 cells at random, is analyzed with the CASP analysis software for every group.Test repeats twice.
4, the statistical study of comet graphical analysis and data
Choose comet head DNA number percent (HDNA%), afterbody DNA number percent (TDNA%), comet total length (CL), tail long (TL), tail square (TM) and Olive tail square (OTM) as analysis indexes.
Statistical procedures: These parameters is carried out contrast in twos between one-way analysis of variance and group with the SPSS13.0 statistical software.
Claims (7)
1. a gel paving method by using single cell gel electrophoresis comprises the steps:
(1) with the microslide of cleaning in 0.2%～1.0% normal fusing point agarose solution after the submergence, 37 ℃ of dry for standby;
(2) with cell suspension and 1% low melting-point agarose solution with 1: 1 mixing of volume ratio, mixed liquor is dropped on the microslide behind the above-mentioned plated film;
(3) the shop film, at the uniform velocity will be spread film and drag backward near cell suspension with 30～miter angle, and suspension is paved into film like along shop film front end expansion until cell suspension, and shop glue is finished, and 4 ℃ solidify 2～3min.
2. gel paving method by using single cell gel electrophoresis according to claim 1 is characterized in that: 10～40 μ, 1 mixed liquor is dropped on the microslide behind the above-mentioned plated film.
3. gel paving method by using single cell gel electrophoresis according to claim 1 is characterized in that: described shop film is that quality is pliable and tough, waterproof, the avirulence material.
4. gel paving method by using single cell gel electrophoresis according to claim 3 is characterized in that: the shop film is the PVC material.
5. gel paving method by using single cell gel electrophoresis according to claim 4 is characterized in that: described shop film thickness is 0.15～0.35mm.
6. gel paving method by using single cell gel electrophoresis according to claim 5 is characterized in that: the shop film is trapezoidal, wide 1cm and the 0.7cm of being respectively in two ends, and height is 4cm, is used for the vary in size shop system of glue of area.
7. gel paving method by using single cell gel electrophoresis according to claim 5 is characterized in that: the shop film is a strip, and long is 4cm, and wide is 0.7～1cm.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105891305A (en) * | 2016-06-15 | 2016-08-24 | 深圳烟草工业有限责任公司 | Method for detecting biosecurity of cigarette smoke condensates |
| CN107576540A (en) * | 2017-07-12 | 2018-01-12 | 浙江省农业科学院 | A kind of easy comet method |
| CN107576713A (en) * | 2017-07-12 | 2018-01-12 | 浙江省农业科学院 | A kind of method for detecting earthworm coelomocyte DNA damage and its utilization |
| CN109355418A (en) * | 2018-11-16 | 2019-02-19 | 中国检验检疫科学研究院 | A method of identifying whether fresh food fruit passes through quarantine irradiation |
| CN111474231A (en) * | 2019-01-23 | 2020-07-31 | 天津师范大学 | Method for detecting duckweed protoplast DNA damage degree by using single cell gel electrophoresis technology |
| CN112710720A (en) * | 2020-12-17 | 2021-04-27 | 深圳市职业病防治院 | Single cell gel electrophoresis gel spreading method and device |
| CN114397350A (en) * | 2022-01-14 | 2022-04-26 | 中国热带农业科学院热带生物技术研究所 | Preparation method and application of high-concentration agarose electrophoresis gel |
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Cited By (9)
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| CN105891305A (en) * | 2016-06-15 | 2016-08-24 | 深圳烟草工业有限责任公司 | Method for detecting biosecurity of cigarette smoke condensates |
| CN107576540A (en) * | 2017-07-12 | 2018-01-12 | 浙江省农业科学院 | A kind of easy comet method |
| CN107576713A (en) * | 2017-07-12 | 2018-01-12 | 浙江省农业科学院 | A kind of method for detecting earthworm coelomocyte DNA damage and its utilization |
| CN107576713B (en) * | 2017-07-12 | 2019-09-27 | 浙江省农业科学院 | A kind of method and its utilization detecting earthworm coelomocyte DNA damage |
| CN109355418A (en) * | 2018-11-16 | 2019-02-19 | 中国检验检疫科学研究院 | A method of identifying whether fresh food fruit passes through quarantine irradiation |
| CN111474231A (en) * | 2019-01-23 | 2020-07-31 | 天津师范大学 | Method for detecting duckweed protoplast DNA damage degree by using single cell gel electrophoresis technology |
| CN112710720A (en) * | 2020-12-17 | 2021-04-27 | 深圳市职业病防治院 | Single cell gel electrophoresis gel spreading method and device |
| CN114397350A (en) * | 2022-01-14 | 2022-04-26 | 中国热带农业科学院热带生物技术研究所 | Preparation method and application of high-concentration agarose electrophoresis gel |
| CN114397350B (en) * | 2022-01-14 | 2024-04-12 | 中国热带农业科学院热带生物技术研究所 | Preparation method and application of high-concentration agarose electrophoresis gel |
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Application publication date: 20101020 |