CN102424855A - Method for carrying out silver staining on DNA (deoxyribonucleic acid) in polyacrylamide gel - Google Patents
Method for carrying out silver staining on DNA (deoxyribonucleic acid) in polyacrylamide gel Download PDFInfo
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- CN102424855A CN102424855A CN2011104494416A CN201110449441A CN102424855A CN 102424855 A CN102424855 A CN 102424855A CN 2011104494416 A CN2011104494416 A CN 2011104494416A CN 201110449441 A CN201110449441 A CN 201110449441A CN 102424855 A CN102424855 A CN 102424855A
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Abstract
The invention relates to a method for carrying out silver staining on DNA (deoxyribonucleic acid) in polyacrylamide gel. The method comprises the following steps: after an electrophoresis process is completed, soaking polyacrylamide gel into a staining solution (comprising 8% of ethanol, 0.6% of a nitric acid and 0.01-0.08% of silver nitrate) for 10-30 minutes; then, carrying out color development on the polyacrylamide gel for 10 minutes by using a color-developing solution (comprising 1.5% of NaOH, 0.4% of formaldehyde and 90 mg/L of sodium borohydride or potassium borohydride); and finally, carrying out terminating for 2 minutes by using a 1% nitric acid. In traditional methods and many improved methods, because the concentration of silver nitrate is high (0.1-0.2%) in the process of staining, an operation of gel washing is required to be performed after a silver staining operation is performed so as to remove silver ions in the gel; but because the thickness and size of the gel are different, the gel washing time is difficult to control. Compared with traditional methods and many improved methods, by using the method provided by the invention, the concentration of silver nitrate is lower, the concentration of silver nitrate is only 0.01% in the process of micro-gel staining, and the concentration of silver nitrate is only 0.05% in the process of macro-gel staining, so that silver nitrate is saved, and a washing step performed after silver staining is saved, therefore, the method has the characteristics of simpleness, convenience and economy; and the repeatability of silver staining is greatly increased while a staining effect is guaranteed.
Description
Technical field
The present invention relates to the silver staining method of DNA in a kind of polyacrylamide gel, belong to the biomacromolecule dyeing process of biology field.
Background technology
Argentation was set up in 1979; Like Switaer at document 1 (Switer R.C.; Merrill C.R.; Shifrins.Ahighly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels.Anal Biochem, 1979, studied with silver staining method in 98:231) the polyacrylamide gel protein electrophoresis has been dyeed.This method had been used in the detection of nucleic acid afterwards; Argentation has improved the sensitivity that detects greatly; Promoted the research of biomacromolecule 26S Proteasome Structure and Function; Be widely used in nucleic acid at present and separated, particularly in AFLP (amplified fragment length polymorphism), SSR (simple repeat sequence), SNP (single-nucleotide polymorphism) equimolecular marker research, had irreplaceable effect with proteinic.
The ultimate principle of silver staining method is with stationary liquid nucleic acid to be fixed on the gel earlier, makes the silver ions mortise with it in the silver-colored stain then, again through reductive agent with silver ion reduction, thereby coupling reaction has taken place, make title product visible.Argentation is less demanding to reagent, and stationary liquid and staining fluid can reuse, and has reduced experimentation cost; The agents useful for same safety and stability; The hazardness that health caused to the operator is very little, and the gel after silver dyes can prolonged preservation, more helps the retrospective analysis to test-results.Dye used argentum reagent according to silver; Silver dyes and can be divided into silver nitrate method staining that exists with acid form and the silver-colored ammonia staining that exists with alkaline form; Wherein more general with silver nitrate method staining; Argentation has very high sensitivity, can make strand and double-strandednucleic acid painted, therefore obtains the favor of vast researcher.
Argentation commonly used at present is Bassam method (Bassam B J; Caetano A G, Gresshoff P M.Fast and sensitive silver staining of DNA in polyacrylamide gels [J] .Analytical Biochemistry, 1991; 196:80-83.) and Sanguinetti method (Sanguinetti C J; Dias N E, Simpson A J.Rapid silver staining and recovery of PCR products separated on polyacrylamide gels [J] .Biotechniques, 1994; 17 (5): 914-921.), but process is more loaded down with trivial details.In recent years; Many researchers has proposed many improved argentations according to oneself practice, but each defectiveness all, the operation steps that has is too many, consuming time longer; The longest report that has needs 22 hours; What have saves time, but circulation ratio is relatively poor, and the painted result of twice operation of same sample is different fully.
In traditional and the many improvement silver staining methods, because silver nitrate concentration higher (0.1～0.2%) during dyeing all will be washed glue, to remove the silver ions in the gel after silver dyes.But since the thickness of glue with vary in size, wash the glue time and be difficult to control.Wash the glue overlong time, then can make the resolving power of film that obtains low the silver ions flush away that is adsorbed on the DNA, band be fuzzy; It is too short to wash the glue time, can make that then the silver ions washing on the glue face is insufficient, and after through colour developing, the film background is deepened.
Summary of the invention
To the deficiency that existing silver staining method exists, the purpose of this invention is to provide the silver staining method of DNA in the high polyacrylamide gel of a kind of easy, economy and success ratio.
For realizing above-mentioned purpose, solution of the present invention is following:
The silver staining method of DNA in a kind of polyacrylamide gel; It is characterized in that; After electrophoresis finishes; Soak polyacrylamide gel 30min with staining fluid (8% ethanol, 0.6% nitric acid and 0.01%～0.08% Silver Nitrate), with colour developing liquid (1.5%NaOH, 0.4% formaldehyde and 90mg/L Peng Qinghuana or POTASSIUM BOROHYDRIDE 97MIN) colour developing 10min, stop 2min with 1% nitric acid at last then.
Described staining fluid consists of: 8% ethanol, 0.6% nitric acid, 0.01%～0.08% Silver Nitrate.
Described colour developing liquid consists of: 1.5%NaOH, 0.4% formaldehyde, 90mg/L Peng Qinghuana or POTASSIUM BOROHYDRIDE 97MIN).
Method of the present invention is optimized silver nitrate concentration, and is lower than the silver nitrate concentration of traditional method and many modification methods, and its concentration has only 0.01% when dying little glue; Its concentration has only 0.05% when dying big glue, practices thrift Silver Nitrate, saves the step of washing glue after silver dyes; Finally set up a kind of silver staining method of improvement, when ensureing detection sensitivity, controlled the dyeing background of gel effectively; Step is simple, operation easily, and the success ratio of experiment is high.
Description of drawings
Fig. 1 is that yam micro-satellite primers P03, P04, P05 and P06 carry out amplification PCR products 6% denaturing polyacrylamide gel electrophoresis and argentation detection figure of the present invention to three parents of yam (purple skin drinamyl, the Bai Pibai heart, white skin drinamyl) and five F1 gene pools (white skin red heart, the Bai Pibai heart, white skin drinamyl, the white heart of purple skin, purple skin drinamyl) respectively.
Fig. 2 is that yam micro-satellite primers P01, P02, P03 and P04 carry out amplification PCR products 6% denaturing polyacrylamide gel electrophoresis and Bassam argentation detection figure to three parents of yam (purple skin drinamyl, the Bai Pibai heart, white skin drinamyl) and five F1 gene pools (white skin red heart, the Bai Pibai heart, white skin drinamyl, the white heart of purple skin, purple skin drinamyl) respectively.
Embodiment
The preparation and the electrophoresis of embodiment 1:6% denaturing polyacrylamide gel.
Table 1 gel formula
| Little glue (120mm * 100mm * 1mm) | Big glue (500mm * 340mm * 2mm) | |
| 40% SEPIGEL 305 | ?6mL | 90mL |
| Urea | ?16.8g | 252g |
| 50x?TBE | ?0.8mL | 12mL |
| Ammonium persulphate | ?14mg | 150mg |
| TEMED | ?28μL | 400μL |
| Deionized water | ?19mL | 284mL |
| Final volume | ?40mL | 600mL |
PCR product and sex change damping fluid (98% methane amide with yam SSR; 10mmol/L EDTA; 0.25% tetrabromophenol sulfonphthalein) equal-volume mixes, and behind 95 ℃ of water-bath sex change 5min, changes in the ice-water bath immediately and cools off; The back adds 1 μ L mixed solution in the appearance hole on each in little glue, in big glue, add 2 μ L mixed solutions in the appearance hole on each.Little glue 250V electrophoresis 2h, big glue 1500V electrophoresis 2.5h.
Embodiment 2: little glue dyeing condition is optimized.
The preparation silver nitrate concentration is respectively each 100mL of staining fluid of 0.007%, 0.009%, 0.011%, 0.013%, 0.015%, 0.017% and 0.019%, soaks little glue 20min, with colour developing liquid colour developing 20min, stops 2min with 1% nitric acid at last then.The result shows, with the staining fluid that contains 0.015% Silver Nitrate little glue is carried out silver and dyes, and can obtain the quality height, and background colour is low, resolving power is high, and band is film clearly.
Dyeing time is controlled between the 20-30min, just can obtain the high film of quality.
Developing time is respectively 15min, 20min, 25min, 30min.Analysis through to the result is compared, and can find out that developing time is controlled between the 20-25min, can obtain the quality height, and background colour is low, resolving power is high, and band is film clearly.
Compare three kinds of colour developings liquid A (1.5%NaOH and 0.4% formaldehyde), B (1.5%NaOH, 0.4% formaldehyde and 90mg/L NaBH4) and C (0.65%Na
2CO
3, 1.3%NaOH and 0.4% formaldehyde) effect, show 1.5%NaOH, 0.4% formaldehyde and 90mg/LNaBH
4During for colour developing liquid, the gained film quality is high, and background colour is low, resolving power is high, and band is clear.
Embodiment 3: big glue dyeing condition is optimized.
The silver that little glue is obtained dyes the result to be tried out when big glue, because the area of little glue, thickness and big glue differ greatly, and big glue carries out having only when silver dyes a glue face to contact with staining reagent, therefore, big glue being carried out silver when dying, adjust silver nitrate concentration.The preparation silver nitrate concentration is respectively each 2000mL of staining fluid of 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07% and 0.08%, soaks big glue 40min, with colour developing liquid colour developing 20min, stops 2min with 1% nitric acid at last then.The result shows, with the staining fluid that contains 0.05% Silver Nitrate big glue is carried out silver and dyes, and can obtain the quality height, and background colour is low, resolving power is high, and band is film clearly.
Embodiment 4:Bassam argentation and argentation of the present invention are relatively
Table 2 Bassam argentation and argentation of the present invention are relatively
Press table 2 operation steps relatively Bassam argentation and argentation of the present invention, the result explain that method of the present invention is suitable with the Bassam method, but method of the present invention is easier and economical shown in Fig. 1 (argentation of the present invention) and Fig. 2 (Bassam method).
Claims (3)
1. the silver staining method of DNA in the polyacrylamide gel; It is characterized in that; After electrophoresis finishes; Soak polyacrylamide gel 10-30min with staining fluid (8% ethanol, 0.6% nitric acid and 0.01%～0.08% Silver Nitrate), with colour developing liquid (1.5%NaOH, 0.4% formaldehyde and 90mg/L Peng Qinghuana or POTASSIUM BOROHYDRIDE 97MIN) colour developing 10min, stop 2min with 1% nitric acid at last then.
2. method according to claim 1 is characterized in that staining fluid consists of: 8% ethanol, 0.6% nitric acid, 0.01%～0.08% Silver Nitrate.
3. method according to claim 1 is characterized in that: colour developing liquid consists of: 1.5%NaOH, 0.4% formaldehyde, 90mg/L Peng Qinghuana or POTASSIUM BOROHYDRIDE 97MIN).
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106596232A (en) * | 2016-12-13 | 2017-04-26 | 广州大学 | Silver staining kit for detecting DNA in polyacrylamide gel and application of silver staining kit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101354348A (en) * | 2008-08-28 | 2009-01-28 | 云南省农业科学院甘蔗研究所 | Ultra-rapid silver dye detecting method of sugarcane molecular marker PAGE gel |
| CN101634615A (en) * | 2008-07-25 | 2010-01-27 | 中国水产科学研究院长江水产研究所 | Silver staining method of polyacrylamide gel |
| CN101865798A (en) * | 2010-06-12 | 2010-10-20 | 西北农林科技大学 | DNA silver staining method in polyacrylamide gel electrophoresis |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101634615A (en) * | 2008-07-25 | 2010-01-27 | 中国水产科学研究院长江水产研究所 | Silver staining method of polyacrylamide gel |
| CN101354348A (en) * | 2008-08-28 | 2009-01-28 | 云南省农业科学院甘蔗研究所 | Ultra-rapid silver dye detecting method of sugarcane molecular marker PAGE gel |
| CN101865798A (en) * | 2010-06-12 | 2010-10-20 | 西北农林科技大学 | DNA silver staining method in polyacrylamide gel electrophoresis |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106596232A (en) * | 2016-12-13 | 2017-04-26 | 广州大学 | Silver staining kit for detecting DNA in polyacrylamide gel and application of silver staining kit |
| WO2018107879A1 (en) * | 2016-12-13 | 2018-06-21 | 广州大学 | Silver staining kit for detecting dna in polyacrylamide gel, and use thereof |
| CN106596232B (en) * | 2016-12-13 | 2019-04-30 | 广州大学 | The silver staining kit of DNA and its application in a kind of detection polyacrylamide gel |
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