CN104007161A - Polyacrylamide gel solution for microsatellite marker polymorphism detection and silver staining method - Google Patents
Polyacrylamide gel solution for microsatellite marker polymorphism detection and silver staining method Download PDFInfo
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- CN104007161A CN104007161A CN201410199761.4A CN201410199761A CN104007161A CN 104007161 A CN104007161 A CN 104007161A CN 201410199761 A CN201410199761 A CN 201410199761A CN 104007161 A CN104007161 A CN 104007161A
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Abstract
The invention discloses a polyacrylamide gel (PAG) solution for microsatellite marker (also known as simple sequence repeats or SSR) polymorphism detection. The polyacrylamide gel solution includes a polyacrylamide gel mother liquor, 5*TBE, a 10% APS and TEMED, diluting is carried out by adopting distilled water until the volume of the polyacrylamide gel mother liquor accounts for 21-22% of the total volume, and the amount of the 5*TBE is the same as that of the polyacrylamide gel mother liquor. When the polyacrylamide gel solution is used, PAG has less damage during an oscillation developing process, PAG splicing is avoided, the experimental error is reduced, and the accuracy of results is guaranteed.
Description
Technical field
The present invention relates to gel reagents and using method thereof used in a kind of SSR testing process, belong to biological technical field.
Background technology
Micro-satellite (SSR), claim again simple sequence to repeat (Simple Sequence Repeats, SSR), the section of DNA that the base unit being comprised of 1～6 nucleotide repeatedly forms, be distributed widely in genomic diverse location, length is generally below 200bp.Because the multiplicity of recurring unit is variability and quantity is abundant between individuality, so the application of microsatellite marker is very extensive.Microsatellite locus is conventionally by pcr amplification, and amplified production detects by electrophoretic analysis and according to size separation allele.
In SSR pleiomorphism detecting method, PAGE polyacrylamide gel electrophoresis (polyacrylamide gel electrophoresis) is a kind of method of widespread use, adopts silver staining method to dye to polyacrylamide gel electrophoresis.
The ultimate principle of silver staining method is first with immobile liquid, nucleic acid to be fixed on gel, then makes silver ion in silver-colored stain strong bonded with it, then by reductive agent by silver ion reduction, thereby there is chromogenic reaction, make target product visible.Argentation is less demanding to reagent, and immobile liquid and dyeing liquor can be reused, reduced experimentation cost, agents useful for same safety and stability, the harmfulness that operator's health is caused is very micro-, gel after silver dyes can be preserved for a long time, is more conducive to the retrospective analysis to test findings, and therefore the silver staining method based on PAGE is widely applied at biological technical field.
Polyacrylamide gel (Polyacrylamide Gel, PAG) be by acrylamide monomer (Acrylamide, be called for short Acr) and crosslinking chemical N1, N1-methylene diacrylamide (N, N-methylene bisacrylamide, is called for short Bis) aliphatic long-chain of the amide-containing side chain that is polymerized under the effect of catalyzer, two adjacent chains get up by methene bridge interlinkage, chain is crisscross, forms the gel of tridimensional network.The size in PAG aperture is mainly determined by the gel strength of Acr and these two kinds of monomers of Bis.Gel strength can change in 3%～30%.In microsatellite marker exploitation and testing process, generally select the concentration of Acr/Bis:20-40.Under this concentration, PAG is completely transparent and flexible, but in process color, PAG glue is easily cracked, when taking pictures, need to carry out " picture mosaic ", and when data analysis, the PAG of cracked rear splicing can increase the error of result; Meanwhile, existing detection method also exists PAG color after colour developing dark for SSR, the band problem such as be not easily distinguishable.
Summary of the invention
Defect for prior art, the invention discloses a kind of polyacrylamide gel solution detecting for Polymorphism of Microsatellite Markers and corresponding silver staining method, adopt gel of the present invention to carry out silver and dye colour developing, PAG is in vibration process color, damaged less, avoid PAG splicing, reduced experimental error, guaranteed the accuracy of result.
For achieving the above object, the present invention is achieved through the following technical solutions:
A kind of polyacrylamide gel solution detecting for Polymorphism of Microsatellite Markers, comprise polyacrylamide gel mother liquor, 5 * TBE, 10%APS (ammonium persulfate), TEMED (tetramethylethylenediamine), with distilled water diluting, to polyacrylamide gel mother liquor volume, account for the 21-22% of cumulative volume, wherein 5 * TBE consumption is identical with polyacrylamide gel mother liquor.
In above-mentioned, each component content all calculates with volume ratio.
In the present invention, described polyacrylamide gel mother liquid concentration is 20-40%, and optimization polypropylene acrylamide gel mother liquid concentration is 30%.
It is identical that above-mentioned PAG mother liquor used and common PAG mother liquor form, by acrylamide and methylene diacrylamide according to a certain percentage (normally 20-30:1) with distilled water, be settled to desired concn.For example the compound method of 30% polyacrylamide gel mother liquor is 580g acrylamide, 20g methylene diacrylamide, and distilled water is settled to 2L, the preservation of spending the night of 37 ℃ of constant temperature.
In the present invention, 10%APS (mass body volume concentrations), TEMED adopt conventional amount used of the prior art, normally 10%APS consumption (volume) accounts for the 0.85-0.95% of gel solution cumulative volume, and TEMED consumption is 1/10 of 10%APS (volume).
Wherein, 5 * TBE can adopt this type of commercially available damping fluid, consist of those skilled in the art extensively known to, be to adopt Tris alkali 54g typical composition, boric acid 27.5g, Na2EDTA.2H2O4.65g, is dissolved in 750ml distilled water, add distilled water again and be settled to 1000ml, commercial preparation adopts this composition mostly at present.
On the basis of the above, the invention also discloses the silver staining method of the polyacrylamide gel DNA detecting for Polymorphism of Microsatellite Markers, after electrophoresis finishes, with PAG dyeing liquor, soak polyacrylamide gel 10-30min claimed in claim 1, then with PAG nitrite ion colour developing 5-10min, finally with stop buffer, stop 2-3min; Wherein in PAG nitrite ion, the content of formaldehyde is 0.2%.
In said method, the formaldehyde by specific consumption is as reductive agent, effectively accelerates the reduction with the Ag+ of protein bound, based on polyacrylamide gel solution of the present invention, formaldehyde consumption is significantly lower than classic method, while developing the color for PAG, of light color, band is clear, and accuracy in detection is higher.
Wherein, PAG dyeing liquor used consists of: 10% ethanol (volumetric concentration), 0.5% glacial acetic acid (volumetric concentration), 0.1%～0.2% silver nitrate (mass body volume concentrations).
Preferably, silver nitrate consumption is 0.2%.
Wherein, PAG nitrite ion forms: 0.04% sodium carbonate (mass body volume concentrations), 2% NaOH (mass body volume concentrations), 0.2% formaldehyde (volumetric concentration).
By gel solution of the present invention and silver staining method thereof, there is superior detection sensitivity, the dyeing background of effectively controlling gel makes it more clear, and step is simple, easily operation, the success ratio of experiment is high.
Accompanying drawing explanation
Fig. 1 is the testing result figure that wide mouthful of schizothoracin K-9SSR primer polymorphic detection adopts polyacrylamide gel electrophoresis of the present invention and argentation;
Fig. 2 is for adopting polyacrylamide gel solution and the testing result figure of argentation to wide mouthful of schizothoracin K-9SSR primer polymorphism in conventional art.
Embodiment
In the following embodiments, the present invention's raw material used:
30%PAG mother liquor: 580g acrylamide, 20g methylene diacrylamide, distilled water is settled to 2L, and 37 ℃ of constant temperature spend the night;
PAG dyeing liquor: AgNO31g, absolute ethyl alcohol 50ml, glacial acetic acid 2.5ml, adds distilled water and is settled to 500ml, keeps in Dark Place;
PAG nitrite ion: sodium carbonate 0.2g, NaOH 10g, adds distilled water and is settled to 500ml, preserves at 4 ℃, adds the 1ml formaldehyde supporting with PAG nitrite ion before use, stand-by;
5 * TBE:Tris54g, boric acid 27.5g, Na2EDTA.2H2O4.65g, is dissolved in 750ml distilled water, then adds distilled water and be settled to 1000ml.
Based on above-mentioned raw materials, prepared following sample:
| Reagent | Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 | Embodiment 6 | Embodiment 7 |
| 30％PAG | 8ml | 16ml | 24ml | 32ml | 40ml | 48ml | 56ml |
| H 2O | 20.5ml | 41ml | 61.5ml | 82ml | 102.5ml | 123ml | 143.5ml |
| 5×TBE | 8ml | 16ml | 24ml | 32ml | 40ml | 48ml | 56ml |
[0029]?
| 10％APS | 325ul | 650ul | 975ul | 1300ul | 1625ul | 1950ul | 2275ul |
| TEMED | 32.5ul | 65ul | 97.5ul | 130ul | 162.5ul | 195ul | 227.5ul |
| Cumulative volume | ≈37ml | ≈74ml | ≈111ml | ≈148ml | ≈185ml | ≈222ml | ≈259ml |
Comparative example, adopts following raw material:
30%PAG mother liquor: 580g acrylamide, 20g methylene diacrylamide, distilled water is settled to 2L, and 37 ℃ of constant temperature spend the night;
PAG dyeing liquor: AgNO31g, absolute ethyl alcohol 50ml, glacial acetic acid 2.5ml, adds distilled water and is settled to 500ml, keeps in Dark Place;
PAG nitrite ion: sodium carbonate 0.2g, NaOH 10g, adds distilled water and is settled to 500ml, preserves at 4 ℃, adds 2ml formaldehyde before use, stand-by;
5 * TBE:Tris54g, boric acid 27.5g, Na2EDTA.2H2O4.65g, is dissolved in 750ml distilled water, then adds distilled water and be settled to 1000ml.
Based on above-mentioned raw materials, prepare following comparative example's sample:
| Reagent | Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 | Comparative example 5 | Comparative example 6 | Comparative example 7 |
| 30％PAG | 7ml | 14ml | 21ml | 28ml | 35ml | 42ml | 49ml |
| H 2O | 20.5ml | 41ml | 61.5ml | 82ml | 102.5ml | 123ml | 143.5ml |
| 5×TBE | 7ml | 14ml | 21ml | 28ml | 35ml | 42ml | 49ml |
| 10％APS | 325ul | 650ul | 975ul | 1300ul | 1625ul | 1950ul | 2275ul |
| TEMED | 32.5ul | 65ul | 97.5ul | 130ul | 162.5ul | 195ul | 227.5ul |
| Cumulative volume | ≈35ml | ≈70ml | ≈105ml | ≈140ml | ≈175ml | ≈210ml | ≈245ml |
Based on above-described embodiment, carried out control test
The polyacrylamide gel solution of getting respectively above-described embodiment 1 and comparative example 1 detects through the product of pcr amplification wide mouthful of identical schizothoracin K-9SSR, as shown in accompanying drawing 1,2, as can be seen from the figure, it is high that the present invention can obtain quality, background colour is low, resolution is high, and band is film clearly.
Claims (6)
1. the polyacrylamide gel solution detecting for Polymorphism of Microsatellite Markers, it is characterized in that comprising polyacrylamide gel mother liquor, 5 * TBE, 10%APS, TEMED, with distilled water diluting, to polyacrylamide gel mother liquor volume, account for the 21-22% of cumulative volume, wherein 5 * TBE consumption is identical with polyacrylamide gel mother liquor.
2. polyacrylamide gel solution according to claim 1, is characterized in that described polyacrylamide gel mother liquid concentration is 20-40%.
3. polyacrylamide gel solution according to claim 1, is characterized in that described polyacrylamide gel mother liquid concentration is 30%.
4. the silver staining method of a polyacrylamide gel DNA who detects for Polymorphism of Microsatellite Markers, it is characterized in that after electrophoresis finishes, with PAG dyeing liquor, soak polyacrylamide gel 10-30min claimed in claim 1, then with PAG nitrite ion colour developing 5-10min, finally with stop buffer, stop 2-3min; Wherein in PAG nitrite ion, the content of formaldehyde is 0.2%.
5. method according to claim 4, is characterized in that PAG dyeing liquor consists of: 10% ethanol, 0.5% glacial acetic acid, 0.1%～0.2% silver nitrate.
6. method according to claim 4, is characterized in that PAG nitrite ion consists of: 0.04% sodium carbonate, 2% NaOH, 0.2% formaldehyde.
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| CN107860812A (en) * | 2017-09-19 | 2018-03-30 | 海南热带海洋学院 | A kind of kit polyacrylamide gel electrophoresis glue and the volley of rifle fire intersect high flux spotting methods |
| WO2018107879A1 (en) * | 2016-12-13 | 2018-06-21 | 广州大学 | Silver staining kit for detecting dna in polyacrylamide gel, and use thereof |
| CN109374715A (en) * | 2018-09-29 | 2019-02-22 | 山东省农业科学院玉米研究所（山东省农业科学院玉米工程技术研究中心） | A kind of molecular markers for identification polyacrylamide gel preparation method |
| CN111972324A (en) * | 2020-09-10 | 2020-11-24 | 四川律贝生物科技有限公司 | Breeding and artificial propagation method of schizothorax prenanti |
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Application publication date: 20140827 |