CN101477001A - DNA silver staining method - Google Patents
DNA silver staining method Download PDFInfo
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- CN101477001A CN101477001A CNA2009100640562A CN200910064056A CN101477001A CN 101477001 A CN101477001 A CN 101477001A CN A2009100640562 A CNA2009100640562 A CN A2009100640562A CN 200910064056 A CN200910064056 A CN 200910064056A CN 101477001 A CN101477001 A CN 101477001A
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Abstract
The invention discloses a DNA silver staining method. The method comprises the following steps: (1) cutting off power supply after finishing electrophoresis, pouring a buffer solution out from an electrophoresis tank, then taking off a gel plate, putting the rubber plate into a porcelain dish filled with distilled water, and rinsing the gel plate by the distilled water for 2 to 3 times; (2) carrying out silver staining: adding a staining agent to carry out silver staining, shaking ceaselessly, staining for 8 to 12 minutes, and then rinsing by the distilled water for 2 to 3 times; (3) carrying out developing: adding a developing agent to carry out color development, shaking ceaselessly, and developing for 8 to 12 min; (4) rinsing by the distilled water for 2 to 3 times; and (5) carrying out gel shooting: spreading gelatin onto a X-ray film viewer to take a photo. The method integrates the fixing step and the staining step, adopts ethanol to fix, and overcomes the defects that the belt color is lighter when acetic acid is used for fixing, and the acetic acid has penetrating odor, causticity and so on in the prior sliver staining method. Additionally, the method does not need use ethanol and/ or acetic acid to stop developing color, omits the color development stopping step, and solves the problems of penetrating odor and causticity brought by the acetic acid and the like.
Description
Technical field
The present invention relates to a kind of DNA silver staining method, relate in particular to a kind of silver and dye and coloration method DNA in the denaturing polyacrylamide gel (PAGE).
Background technology
Polyacrylamide gel electrophoresis (Polyacrylamide gel electrophoresis, PAGE) be the method a kind of commonly used of analysing protein and nucleic acid samples, because it has higher resolution, in the research of molecular biology and related discipline, used widely, especially used more at aspects such as AFLP, SSR, SNP equimolecular marker research and genetic map constructions.The method many behind the electrophoresis to showing of sample, development process is mainly taked in the detection of target product, the development process that adopts has following several substantially at present: (1) isotope autoradiography, (2) fluorochrome label method, (3) ethidium bromide staining method, wherein, the isotope autoradiography has highly sensitive, advantages such as good reliability, but must use isotope labeling, gel shifts, steps such as autography, operating process is more loaded down with trivial details, and the operator subjects to the danger of isotope irradiation, need add special equipment and safeguard procedures, thereby certain degree of difficulty is arranged in applying; Isotope is to people's harmfulness in the method (1) though the fluorochrome label method has overcome, and the while has also been reduced the sensitivity that detects, and operating process is loaded down with trivial details equally, and experimentation cost is also higher, needs equipment such as fluorescent microscope; Ethidium bromide (EB) decoration method is convenient and easy, but polyacrylamide has quenching effect to the fluorescence of ethidium bromide EB, greatly reduce ethidium bromide staining sensitivity, DNA band less than 10ng is difficult to detect, in addition, often require the amount of product bigger, and EB has carcinogenicity, make the operation of gel and the processing complexity that becomes.
Adopt argentation, can overcome the shortcoming of above three kinds of common gel staining methods effectively, the ultimate principle of argentation is: with immobile liquid nucleic acid is fixed on the gel earlier, make the silver ion strong bonded with it in the silver-colored dye liquor then, again by reductive agent with silver ion reduction, thereby chromogenic reaction has taken place, made target product can see with naked eyes.Argentation is not high relatively to the requirement of reagent, general homemade analytical reagent can meet the demands, and immobile liquid and dyeing liquor can reuse, and has reduced experimental cost, gel after silver dyes can long preservation, more helps the retrospective analysis to experimental result.On present molecular biology research, has promotional value widely.Commonly used is the argentation of Bassam (1991) and Sanguinetti (1994) at present, and Many researchers has proposed many improved argentations (Rder et al., 1998 according to the practice of oneself simultaneously; Xu Shaobin etc. 2002), be mainly used in polyacrylamide gel electrophoresis dyeing, also be used for Ago-Gel dyeing, its remolding sensitivity EB is high 200 times, but silver dyeing back DNA should not reclaim.Argentation is the highest a kind of decoration method of sensitivity up to now, and existing argentation mostly has following shortcoming: (1) may cause very high background especially under the pure inadequately situation of water; (2) wasting time and energy (needs 1～2h); (3) expense costliness; (4) need the poisonous material of contact more.At present, Chang Yong silver staining method mainly comprises following several steps: (1) pre-service is with fixing; (2) dyeing; (3) colour developing; (4) cessation reaction.Whole process is more loaded down with trivial details, need use number of chemical reagent such as deionized water, ethanol, nitric acid, silver nitrate, sodium carbonate, formaldehyde, sodium thiosulfate and acetate.Existing silver staining method all needs to adopt ethanol or/and acetate comes color development stopping, and institute's reagent of using is more, and operation steps is comparatively complicated, and correspondingly developing time is longer.When dyeing at every turn, currently used silver staining method need join immobile liquid, developer solution and dyeing liquor, dyeing liquor also important affair carries out precooling etc. earlier, simultaneously very fast at developing process later stage developing powder, timely cessation reaction when it requires banding pattern to show, as hold improper, prolongation along with dyeing time, can make glue dye more black and more black, reduce the contrast of DNA banding pattern and background, Color is relatively poor.
Summary of the invention
The object of the present invention is to provide a kind of running time short, use the few silver staining method of reagent DNA in the denaturing polyacrylamide gel (PAGE).
To achieve these goals, technical program of the present invention lies in having adopted a kind of DNA silver staining method, the step of this method is as follows:
(1) electrophoresis is cut off the electricity supply after finishing, and pours out damping fluid from electrophoresis tank, takes off offset plate then, puts into the porcelain dish that distilled water is housed, with distilled water rinsing 2-3 times;
(2) silver dyes: the adding coloring agent carries out silver and dyes, and does not stop to shake with hand or shaking table, dyes 8-12 minutes, uses distilled water rinsing 2-3 times then;
(3) develop: add developer and develop the color, and do not stop to shake, developed 8-12 minutes with hand or shaking table, to the purpose band clear till;
(4) with distilled water rinsing 2-3 times;
(5) gel is photographed: gel is tiled on the X line film viewbox takes a picture.
Coloring agent in the step (2) is by 0.1-0.2% AgNO
3Form with 10% absolute ethyl alcohol.
Preferably, the coloring agent in the step (2) is by 0.2% AgNO
3Form with 10% absolute ethyl alcohol, behind 0.2% cma staining, the banding pattern colour developing is darker, faster, and sensitivity is higher; The absolute ethyl alcohol of adding 10% in the dyeing course, follow-up process color is fast, not drift of band, banding pattern is neat.
Developer in the step (3) is made up of 2-3% NaOH and 0.2-0.4% formaldehyde.
Preferably, the developer in the step (3) is made up of 2-3% NaOH and 0.3% formaldehyde.
Method of the present invention integrates fixing step and staining procedure, and adopts ethanol to fix, and has avoided adopting in the conventional silver staining method acetate to fix and the band look more shallow that brings, and it has penetrating odor simultaneously, has defectives such as corrosivity; Simultaneously, ethanol and silver nitrate are formulated together, can make follow-up process color fast, not drift of band, and banding pattern is neat.In the developer, the effect of formaldehyde is to be argent with silver ion reduction, and during colour developing, when concentration of formaldehyde was higher, dyeing time shortened, but the jaundice of glue color, the background dirt, and contrast diminishes mutually, and the DNA of different molecular weight can not be developed the color synchronously, influences sensitivity; When concentration is hanged down, not only dyeing time prolongation but also not easy coloring, the present invention preferred 0.3% formaldehyde can obtain hypersensitivity and specific silver dyes the result; NaOH works to provide alkaline environment, promotes the developer development capability in developer.In addition, method of the present invention need not to adopt ethanol or/and acetate comes color development stopping, has saved color development stopping step, has avoided adopting the penetrating odor that acetate brought, and has problems such as corrosivity.
DNA silver staining method of the present invention only need be prepared coloring agent and two kinds of solution of developer, has significantly reduced the kind of solution preparation in the dyeing course, thereby has simplified the colour developing program greatly; And method of the present invention has been saved sodium thiosulfate that development step adopted and the used acetate of color development stopping step that uses in the conventional method in dying the glue process, has reduced the consumption of reagent, has reduced cost; The more important thing is that it is time saving and energy saving, easy and simple to handle, developing process is relatively gentleer, control easily, when gel is put into developer, initial 5min does not have significant change, 2-4min afterwards shows banding pattern gradually, after banding pattern all showed to come, background color was constant substantially, this shows that adopting method of the present invention to develop controls easily.
The concentration of the used NaOH of the present invention is lower, even the proper extension dyeing time can not dye glue blackly yet, the step that therefore need not stop showing after banding pattern shows, is only used the distilled water rinsing, washes developer solution remaining on the glue off and gets final product.
The present invention explores polypropylene amine gel electrophoresis technology colouring method and optimum condition thereof, through repeatedly experiment, a kind of DNA silver staining method has been set up in final research, when increasing substantially detection sensitivity, can control the dyeing background of gel effectively, step is simple, easily operation, whole dyeing course required time short (15-20min), utilize argentation of the present invention that the PAGE gel of grape microsatellite DNA amplified production is dyeed and obtained well-content effect.
In addition, method of the present invention has overcome the deficiency of many non-sex change polyacrylamide gel electrophoresis and argentation, save time easy and good reproducibility, the demonstration and the observation of positive band in the non-sex change polyacrylamide gel have been guaranteed, can reach the purpose of accurate examination and accurate quantification, the positive sample that examination is gone out carries out autotelic order-checking, the fund waste that can avoid blindly order-checking to be brought.
Description of drawings
Fig. 1 dyes design sketch for the silver of silver staining method of the present invention;
Fig. 2 dyes design sketch for the silver of conventional method.
Embodiment
Embodiment 1
The step of DNA silver staining method of the present invention is as follows:
(1) electrophoresis is cut off the electricity supply after finishing, and pours out damping fluid from electrophoresis tank, takes off offset plate then, puts into the porcelain dish that distilled water is housed, with distilled water rinsing 2 times;
(2) silver dyes: add coloring agent and carry out silver and dye, and do not stop to shake with hand or shaking table, dyeed 10 minutes, use the distilled water rinsing then 2 times, wherein coloring agent is by 0.2% AgNO
3(W/V) form with 10% absolute ethyl alcohol (V/V);
(3) develop: add developer and develop the color, and do not stop to shake, developed 10 minutes with hand or shaking table, to the purpose band clear till,, wherein, developer is made up of 3% NaOH (W/V) and 0.3% formaldehyde (V/V);
(4) with distilled water rinsing 2 times;
(5) gel photography: the gel that is tiled on the X line film viewbox is taken a picture with digital camera.
Materials and methods
1.1 material
1.1.1 experiment material
With grape (Vitis vinifera L.) blade is material, uses dH
2O cleans and to remove surface contaminants, blots remained on surface moisture with filter paper, the mark packing, and it is standby to put-20 ℃ of freezing preservations of refrigerator.
1.2 method
1.2.1 the operation steps of modified CTAB method
With reference to the method and the suitably improvement of do of [3] such as Joseph Sambrook, carry out the extraction of genomic DNA.
1.2.2 pcr amplification
The reaction system cumulative volume is 20 μ L, and amplification program is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 1min, 54 ℃ of renaturation 1min, 72 ℃ are extended 1.5min, 30 circulations; 72 ℃ are extended 10min, 4 ℃ of preservations.
1.2.3 6% denaturing polyacrylamide gel electrophoresis detects
1.2.4 show the band method
1.2.4.1
Conventional method
The step of the conventional silver staining method of polyacrylamide gel is as follows:
(1) electrophoresis is cut off the electricity supply after finishing, and pours out damping fluid from electrophoresis tank, takes off offset plate then, gel is taken out from the glass plate put into the porcelain dish that distilled water is housed, with distilled water rinsing 2 times;
(2) fixing: as to add fixedly 30min of immobile liquid (10% glacial acetic acid), use the distilled water rinsing then 2 times;
(3) silver dyes: add dyeing liquor (0.2% AgNO
3, 0.15% formaldehyde) carry out silver and dye 30min, use the distilled water rinsing then 2 times;
(4) develop: add developer solution (30% Na
2CO
3, 0.15% formaldehyde and 2% sodium thiosulfate) develop the color;
(5) when band is clear, acetum is put in the glass plate taking-up, stop about 2 minutes (bubble collapse);
(6) gel photography: gel is taken a picture with digital camera.
2 results and analysis
2.1 different polyacrylamide gel silver staining method silver dye the result
Different polyacrylamide gel silver staining method silver dye and the results are shown in Figure 1-Fig. 2, and Fig. 1 dyes the result for the silver of silver staining method of the present invention, and Fig. 2 dyes the result for the silver of conventional silver staining method.The background that as can be seen from the figure conventional silver staining method silver dyes is more shallow, and purpose fragment and background reflectance are not obvious, brings certain difficulty to accurately declaring type.
The different silver staining method silver of table 1 dye time ratio
Table?1?Comparison?of?time?in?different?sliver-stained?menthod
Embodiment 2
The step of DNA silver staining method of the present invention is as follows:
(1) electrophoresis is cut off the electricity supply after finishing, and pours out damping fluid from electrophoresis tank, takes off offset plate then, puts into the porcelain dish that distilled water is housed, with distilled water rinsing 2 times;
(2) silver dyes: add coloring agent and carry out silver and dye, and do not stop to shake with hand or shaking table, dyeed 9 minutes, use the distilled water rinsing then 2 times, wherein coloring agent is by 0.2% AgNO
3(W/V) form with 10% absolute ethyl alcohol (V/V);
(3) develop: add developer and develop the color, and do not stop to shake, developed 8 minutes with hand or shaking table, to the purpose band clear till, wherein, developer is made up of 2% NaOH (W/V) and 0.4% formaldehyde (V/V);
(4) with distilled water rinsing 3 times;
(5) gel photography: with digital camera to the gel that is tiled on the X line film viewbox being taken a picture with digital camera.
It should be noted last that: above embodiment is only in order to explanation, and unrestricted technical scheme of the present invention, although the present invention is had been described in detail with reference to the foregoing description, those of ordinary skill in the art is to be understood that: still can make amendment or be equal to replacement the present invention, and not breaking away from any modification or partial replacement of the spirit and scope of the present invention, it all should be encompassed in the middle of the claim scope of the present invention.
Claims (6)
1, a kind of DNA silver staining method, it is characterized in that: the step of this method is as follows:
(1) electrophoresis is cut off the electricity supply after finishing, and pours out damping fluid from electrophoresis tank, takes off offset plate then, puts into the porcelain dish that distilled water is housed, with distilled water rinsing 2-3 times;
(2) silver dyes: the adding coloring agent carries out silver and dyes, and does not stop to shake, and dyes 8-12 minutes, uses distilled water rinsing 2-3 times then;
(3) develop: add developer and develop the color, do not stop to shake, developed 8-12 minutes;
(4) with distilled water rinsing 2-3 times;
(5) gel is photographed: gel is tiled on the X line film viewbox takes a picture.
2, DNA silver staining method according to claim 1 is characterized in that: the coloring agent in the step (2) is by 0.1-0.2% AgNO
3Form with 10% absolute ethyl alcohol.
3, DNA silver staining method according to claim 2 is characterized in that: the coloring agent in the step (2) is by 0.2% AgNO
3Form with 10% absolute ethyl alcohol.
4, DNA silver staining method according to claim 1 is characterized in that: the developer in the step (3) is made up of 2-3% NaOH and 0.2-0.4% formaldehyde.
5, DNA silver staining method according to claim 4 is characterized in that: the developer in the step (3) is made up of 2-3% NaOH and 0.3% formaldehyde.
6, according to arbitrary described DNA silver staining method in the claim 1-5, it is characterized in that: in the step (3) develop to the purpose band clear till.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2009100640562A CN101477001A (en) | 2009-01-12 | 2009-01-12 | DNA silver staining method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2009100640562A CN101477001A (en) | 2009-01-12 | 2009-01-12 | DNA silver staining method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101849972A (en) * | 2010-05-07 | 2010-10-06 | 中国人民解放军海军医学研究所 | Banding detection method of Cordyceps sinensis DNA fingerprint high resolution ratio |
| CN102507289A (en) * | 2011-11-15 | 2012-06-20 | 中国农业大学 | Silver staining reagent and staining method thereof |
| CN104007161A (en) * | 2014-05-12 | 2014-08-27 | 塔里木大学 | Polyacrylamide gel solution for microsatellite marker polymorphism detection and silver staining method |
| CN105067751A (en) * | 2015-07-23 | 2015-11-18 | 成都理工大学 | Photocatalysis visualization method for detecting content of silver in ore |
| WO2018107879A1 (en) * | 2016-12-13 | 2018-06-21 | 广州大学 | Silver staining kit for detecting dna in polyacrylamide gel, and use thereof |
-
2009
- 2009-01-12 CN CNA2009100640562A patent/CN101477001A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101849972A (en) * | 2010-05-07 | 2010-10-06 | 中国人民解放军海军医学研究所 | Banding detection method of Cordyceps sinensis DNA fingerprint high resolution ratio |
| CN101849972B (en) * | 2010-05-07 | 2012-05-30 | 中国人民解放军海军医学研究所 | Banding detection method of Cordyceps sinensis DNA fingerprint high resolution ratio |
| CN102507289A (en) * | 2011-11-15 | 2012-06-20 | 中国农业大学 | Silver staining reagent and staining method thereof |
| CN104007161A (en) * | 2014-05-12 | 2014-08-27 | 塔里木大学 | Polyacrylamide gel solution for microsatellite marker polymorphism detection and silver staining method |
| CN105067751A (en) * | 2015-07-23 | 2015-11-18 | 成都理工大学 | Photocatalysis visualization method for detecting content of silver in ore |
| WO2018107879A1 (en) * | 2016-12-13 | 2018-06-21 | 广州大学 | Silver staining kit for detecting dna in polyacrylamide gel, and use thereof |
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Application publication date: 20090708 |