This work was funded by the Guangdong Natural Science Foundation of China (2015A030313500), the Provincial Key International Cooperative Research Platform and the Major Scientific Research Project of Guangdong Higher Education (2015KGJHZ015), the Science and Technology Plan of Guangdong Tobacco Monopoly Administration (201403, 201705), the Science and Technology Plan of Guangdong of China (2016B020201001), the National Innovation Training Project for Undergraduate Students (201711078001). Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. USDA is an equal opportunity provider and employer.

1. Preparation of PCR Products of SSR Markers
<ol>
	<li>Prepare all chemicals and reagents for PCR reactions including template DNA (30 ng/&#181;L), 2&#215; PCR master mix (containing 2&#215; PCR buffer, 0.4 mM of each dNTP, 3 mM of MgCl2, 0.1 U/&#181;L of Taq DNA polymerase and dyes), 10 &#181;M of each forward and reverse primers, and distilled or deionized water (dH2O). 
	NOTE: The SSR markers used in the present study were PT51333 (forward primer sequence: 5 &#39;-GCACCTTTGGTTATCCGACA-3 &...

<ol>
	<li>Jiang, G. L., Anderson, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+markers+and+marker-assisted+breeding+in+plants.">Molecular markers and marker-assisted breeding in plants.</a> Plant breeding from laboratories to fields. , 45-83 (2013).</li><li>Powell, W., Machray, G. C., Provan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymorphism+revealed+by+simple+sequence+repeats.">Polymorphism revealed by simple sequence repeats.</a> Trend Plant Sci. 1, 215-222 (1996).</li><li>Varshney, R. K., Graner, A., Sorrells, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genic+microsatellite+markers+in+plants:+features+and+applications.">Genic microsatellite markers in plants: features and applications.</a> Trend Biotechnol. 23, 48-55 (2005).</li><li>Tuler, A. C., Carrijo, T. T., N&#243;ia, L. R., Ferreira, A., Peixoto, A. L., da Silva Ferreira, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SSR+markers:+a+tool+for+species+identification+in+Psidium+(Myrtaceae).">SSR markers: a tool for species identification in Psidium (Myrtaceae).</a> Mol. Biol. Rep. 42, 1501-1513 (2015).</li><li>Rabilloud, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+protein+silver+staining+in+polyacrylamide+gels:+a+10-year+synthesis.">Mechanisms of protein silver staining in polyacrylamide gels: a 10-year synthesis.</a> Electrophoresis. 11, 785-794 (1990).</li><li>Bassam, B. J., Caetano-Anoll&#233;s, G., Gresshoff, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+sensitive+silver+staining+of+DNA+in+polyacrylamide+gels.">Fast and sensitive silver staining of DNA in polyacrylamide gels.</a> Anal. Biochem. 196, 80-83 (1991).</li><li>Liang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+effective+method+for+silver+staining+of+PCR+products+separated+in+polyacrylamide+gels.">A rapid and effective method for silver staining of PCR products separated in polyacrylamide gels.</a> Electrophoresis. 35, 2520-2523 (2014).</li><li>Switzer, R. C., Merril, C. R., Shifrin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+sensitive+silver+stain+for+detecting+proteins+and+peptides+in+polyacrylamide+gels.">A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels.</a> Anal. Biochem. 98, 231-237 (1979).</li><li>Sanguinetti, C., Dias, N., Simpson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+silver+staining+and+recovery+of+PCR+products+separated+on+polyacrylamide+gels.">Rapid silver staining and recovery of PCR products separated on polyacrylamide gels.</a> Biotechniques. 17, 915-919 (1994).</li><li>Ji, Y., Qu, C., Cao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimal+method+of+DNA+silver+staining+in+polyacrylamide+gels.">An optimal method of DNA silver staining in polyacrylamide gels.</a> Electrophoresis. 28, 1173-1175 (2007).</li><li>Qu, L., Li, X., Wu, G., Yang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+sensitive+method+of+DNA+silver+staining+in+polyacrylamide+gels.">Efficient and sensitive method of DNA silver staining in polyacrylamide gels.</a> Electrophoresis. 26, 99-101 (2005).</li><li>An, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+silver+staining+procedure+for+nucleic+acids+in+polyacrylamide+gels+without+fixation+and+pretreatment.">A silver staining procedure for nucleic acids in polyacrylamide gels without fixation and pretreatment.</a> Anal. Biochem. 391, 77-79 (2009).</li><li>Byun, S. O., Fang, Q., Zhou, H., Hickford, J. G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+effective+method+for+silver-staining+DNA+in+large+numbers+of+polyacrylamide+gels.">An effective method for silver-staining DNA in large numbers of polyacrylamide gels.</a> Anal. Biochem. 385, 174-175 (2009).</li><li>Kumar, M., Kim, S. R., Sharma, P. C., Pareek, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+efficient+way+to+detect+small+polymorphic+bands+in+plants.">Simple and efficient way to detect small polymorphic bands in plants.</a> Genome Data. 5, 218-222 (2015).</li><li>Liu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+simple+and+effective+silver+staining+protocol+for+detection+of+DNA+fragments.">Development of a simple and effective silver staining protocol for detection of DNA fragments.</a> Electrophoresis. 38, 1175-1178 (2017).</li><li>Wang, D., Shi, J., Carlson, S. R., Cregan, P. B., Ward, R. W., Diers, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+low-cost,+high-throughput+polyacrylamide+gel+electrophoresis+system+for+genotyping+with+microsatellite+DNA+markers.">A low-cost, high-throughput polyacrylamide gel electrophoresis system for genotyping with microsatellite DNA markers.</a> Crop Sci. 43, 1828-1832 (2003).</li><li>Echt, C. S., May-Marquardt, P., Hseih, M., Zahorchak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+microsatellite+markers+in+eastern+white+pine.">Characterization of microsatellite markers in eastern white pine.</a> Genome. 39, 1102-1108 (1996).</li></ol>

The washing of gel after impregnation is a critical step. Insufficient washing time and water volume may cause the incomplete removal of impregnation solution on the surface of the plate and the gel, and result in a dark background. The appropriate developing time is another key step, over-development can result in a dark-brown background with low contrast image of DNA fragments. In addition, the impregnation step significantly affects staining efficiency of DNA fragments. Although extending the impregnation time over 5 ...

The development of PCR-based markers has revolutionized the science of plant genetics and breeding1. Simple sequence repeat (SSR) markers are among the most commonly used and most versatile DNA markers. Their broad genome coverage, abundance, genome specificity, and repeatability are some of the merits of SSR markers in addition to their codominant inheritance for the detection of heterozygous genotypes2. Several studies have used SSR markers to investigate genetic diversity, track ancestry, construct genetic linkage maps, and map genes for economically important traits3,4.
PCR products of SSR markers are commonly separated using agarose or polyacrylamide gel electrophoresis and then visualized with silver staining or under UV light after staining with ethidium bromide. Silver staining of DNA fragments in polyacrylamide gels is more sensitive than the other staining methods5,6 and has been widely used to detect DNA fragments such as SSR markers7.
Like many biological laboratory techniques, silver staining of polyacrylamide gels has steadily improved since its first being reported as a fragment visualization technique in 19798. The technique was initially modified for detection of DNA fragments by Bassam et al.6 in 1991 and then improved by Sanguinetti and coworkers9 in 1994. The method has been further optimized in the last few decades6,7,9,10,11,12,13,14,15. However, most of these updated versions of the protocols still have some drawbacks such as high technical demand and long processing time for fixation and mounting6, that limit the application of these protocols7,11. An optimal protocol that combines low-cost with high efficiency of DNA fragment detection is urgently needed for routine application of silver staining in biological research.
In addition, polyacrylamide gel can be divided into denaturing and non-denaturing polyacrylamide gels, and both can be used for the detection of SSR markers using the silver staining method. The effect and resolution of which do not significantly differ, but non-denaturing polyacrylamide gels are easier to process and take less time16.
On the basis of the previous research15, the aim of the current study is to describe an optimized silver staining protocol in detail for quick, easy and low-cost detection of SSR markers in a non-denaturing polyacrylamide gel.

<table>
	<tbody>
		<tr>
			<td>PCR master mix (Green Taq Mix)</td>
			<td>Vazyme Biotech Co. Ltd, China</td>
			<td>#P131-03</td>
			<td></td>
		</tr>
		<tr>
			<td>50-2000 bp DNA Ladder</td>
			<td>Bio-Rad, USA</td>
			<td>#170-8200</td>
			<td></td>
		</tr>
		<tr>
			<td>DL500 DNA marker</td>
			<td>Takara Bio Inc., Japan</td>
			<td>#3590A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sangon Biotech Shanghai, China</td>
			<td>#77-86-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sangon Biotech Shanghai, China</td>
			<td>#10043-35-3</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA-Na2</td>
			<td>Guangzhou Chemical Reagent Factory, China</td>
			<td>#6381-92-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td>Sangon Biotech Shanghai, China</td>
			<td>#79-06-1</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N&#39;-methylene-bis-acrylamide</td>
			<td>Sangon Biotech Shanghai, China</td>
			<td>#110-26-9</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N,N&#39;,N&#39;-Tetramethylethylenediamine</td>
			<td>Sangon Biotech Shanghai, China</td>
			<td>#110-18-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Guangzhou Chemical Reagent Factory, China</td>
			<td>#7727-54-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Bind-silane</td>
			<td>Solarbio Beijing, China</td>
			<td>#B8150</td>
			<td></td>
		</tr>
		<tr>
			<td>AgNO3</td>
			<td>Sinopharm Chemical Reagent Beijing Co.,Ltd, China</td>
			<td>#7761-88-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Tianjin DaMao Chemical Reagent Factory, China</td>
			<td>#50-00-0</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Guangzhou Chemical Reagent Factory, China</td>
			<td>#1310-73-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Guangzhou Chemical Reagent Factory, China</td>
			<td>#64-19-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2CO3</td>
			<td>Tianjin DaMao Chemical Reagent Factory, China</td>
			<td>#497-19-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Guangzhou Chemical Reagent Factory, China</td>
			<td>#64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>HNO3</td>
			<td>Guangzhou Chemical Reagent Factory, China</td>
			<td>#7697-37-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2S2O3.5H2O</td>
			<td>Sinopharm Chemical Reagent Beijing Co.,Ltd, China</td>
			<td>#10102-17-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Eriochrome black T(EBT)</td>
			<td>Tianjin DaMao Chemical Reagent Factory, China</td>
			<td>#1787-61-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic tray</td>
			<td>Shanghai Yi Chen Plastic Co., Ltd, China</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>TS-1 Shaker</td>
			<td>Qilinbeter JiangSu, China</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>BenQ M800 Scanner</td>
			<td>BenQ, China</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>DYY-6C Power supply</td>
			<td>Beijing Liuyi Instrument Factory, China</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>High throughout vertical gel systems, JY-SCZF</td>
			<td>Beijing Tunyi Electrophoresis Co., Ltd, China</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

a fast silver staining protocol enabling simple and efficient detection of ssr markers using a non-denaturing polyacrylamide gel

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver staining is a widely used method for the detection of SSR markers in a polyacrylamide gel. However, conventional protocols for silver staining are technically demanding and time-consuming. Like many other biological laboratory techniques, silver staining protocols have been steadily optimized to improve detection efficiency. Here, we report a simplified silver staining method that significantly reduces reagent costs and enhances the detection resolution and picture clarity. The new method requires two major steps (impregnation and development) and three reagents (silver nitrate, sodium hydroxide, and formaldehyde), and only 7 min of processing for a non-denaturing polyacrylamide gel. Compared to previously reported protocols, this new method is easier, quicker and uses fewer chemical reagents for SSR detection. Therefore, this simple, low-cost, and effective silver staining protocol will benefit genetic mapping and marker-assisted breeding by a quick generation of SSR marker data.

The PCR amplicons were produced using the corresponding SSR primer pairs in flowering Chinese cabbage and tobacco. After electrophoresis, the polyacrylamide gels were stained using the above silver staining protocol, which unambiguously detected the banding patterns of SSR markers (Figure 1).
To compare the detection efficiency of different silver staining protocols, PCR products of SSR markers in t...

Watch this Scientific Journal Video about A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel at JoVE.com

A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel

Here, we report a simple and low-cost silver staining protocol which requires only three reagents and 7 min of processing, and is suitable for fast generation of high-quality SSR data in the genetic analysis.

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver ...

The overall goal of this protocol is to introduce an optimized silver staining method for quick, easy, and low cost detection of SSR markers in non-denaturing polyacrylamide gels. The rapid genotyping of SSR markers can help answer key questions in the genetic research and application field such as genetic diversity analysis and marker assisted selection in molecular breeding programs. The main advantages of this technique is that it's easy to carry out, takes less time and using fewer chemical reagents than other protocols for the detection of SSR markers.This method avoids the fixing, stopping, and several washing steps found in conventional protocols. And only requires the two main steps of impregnation and development. Clear images can be produced using this protocol as no background noise for the unambiguous detection of SSR banding patterns.One can screen approximately 800 DNA samples per day using this technique and it has been used successfully in tobacco and flowering Chinese cabbage research. After preparing PCR products and gel solutions according to the text protocol, use detergent in tap water to wash one set of glass plates and spacers followed by a complete rinsing with distilled water. Set the plates in a plate drying rack to air dry.Disperse one milliliter of Bind-silane solution onto the inner surface of a rectangular glass plate and dry it for five minutes. Then disperse one milliliter of Repel-silane onto the inner surface of a notched glass plate with a swab, and use a tissue paper to wipe off excess solution before allowing the plate to air dry for five minutes. Assemble the glass plates with spacers with the notched plate on top.And use casting clamps to clamp both sides of the assembly. Next, pour an appropriate amount of 6%non-denaturing polyacrylamide gel solution into a beaker for the plate set. Add 20 microliters of TEMED and 200 microliters of fresh 20%APS and gently mix.Immediately and carefully pour the solution into the assembled glass plates along the edge of the notched plate, filling the space almost to the top. And insert the comb. Then add a small volume of gel solution over the comb and allow the gel to set at room temperature for 30 minutes.When the gel is fully polymerized, remove the casting clamps and position the plate set in the electrophoresis tank unit with the notched plate facing toward the upper buffer reservoir. Use large clamps to attach the plate set to the tank unit. Pour one liter of 0.5 X TBE buffer into each of the upper and the lower chambers.Then remove the comb and use a pipette or syringe filled with buffer to thoroughly flush all the wells. To run the polyacrylamide gel, load about one microliter of PCR product into each well. Also, load a DNA ladder on either end.Then fix the safety cover to the upper buffer chamber. Connect the leads to the power supply, matching the color coded red to red, and black to black. Run the gel at a constant voltage of 110 volts until the dye reaches a defined position.Normally, for a approximately 70 minutes. Following electrophoresis, open the valve and drain the buffer from the upper chamber into a large beaker. Detach the side clamps and remove the plate from the apparatus.Then use a spatula to carefully separate the notched glass plate along one side and make sure that the gel remains attached to the other glass plate coated with Bind-silane. Next, prepare one liter of fresh impregnation solution by dissolving 1.5 grams of silver nitrate in one liter of distilled water. Then prepare one liter of fresh development solution by dissolving 10 grams of sodium hydroxide in 900 milliliters of distilled water.Add one milliliter of 37%formaldehyde and use distilled water to adjust to a final volume of one liter. With ample distilled water, carefully rinse the gel and glass plate to remove the electrophoresis buffer. Then place the plate with the gel facing up into a plastic tray and submerge the gel in one liter of impregnation solution.Gently shake the tray on a shaker at 60 RPM for three to four minutes. Transfer the plate out of the impregnation solution into another tray. Then use ample distilled water to rinse the residual impregnation solution off of the surface of the plate and the gel two times for three to five seconds each.The gel wash step after impregnation is critical. Insufficient washing my cause incomplete removal of impregnation solution. And result in dark background.Place the place with the gel facing up into another tray. Submerge the plate in one liter of development solution then gently shake the tray at 50 RPM for approximately three minutes. Monitor the appearance of DNA fragments and stop development when the highest ratio of the signal of DNA fragments to the noise of background is observed.The appropriate developing time is another key step. Overdevelopment can result in a dark brown background with a low contrast image of DNA fragments. Rinse the plate and the gel with ample distilled water twice and use tissue paper to dry the gel plate.Then use a scanner and the appropriate software to scan the gel at 300 DPI and adjust the brightness and contrast to get a clear visualization of the DNA bands. Finally, score the banding pattern of the SSR markers based on the DNA fragment sizes in the image. The PCR amplicons presented here were produced using the corresponding SSR primer pairs in flowering Chinese cabbage and tobacco.After electrophoresis, the polyacrylamide gels were stained using the silver staining protocol demonstrated in this video. Which unambiguously detected the banding patterns of SSR markers. To compare the detection efficiency of different silver staining protocols, PCR products of SSR markers were separated using PAGE, and visualized using five published silver staining protocols and a sixth method demonstrated in this video.The protocol demonstrated here had the lowest background noise and highest contrast of DNA fragments such that it produced the highest picture clarity. Of the six protocols tested, the method demonstrated here takes the least amount of time and requires the fewest number of chemical reagents and process steps. Finally, this figure shows the sensitivity of the protocol as measured using a serial dilution of a 50 to 2000 bp DNA marker on a non-denaturing polyacrylamide gel from 10 nanograms per band in lane one to 9.8 picograms per band of DNA in lane 11.Once mastered, this technique can be done in seven minutes if it is performed properly. While attempting this procedure, it's important to remember to prevent cross-contamination of impregnation and development solutions. After it's development, this technique paved the way for researchers to quickly genotype SSR markers.After watching this video, you should have a good understanding of how to simply and efficient detect SSR markers in a non-denaturing polyacrylamide gel using silver staining. Don't forget that working with chemical reagents can be extremely hazardous and precautions such as gloves, should always be worn while performing this procedure.

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10.6K visualizações.  Guangzhou University. The overall goal of this protocol is to introduce an optimized silver staining method for quick, easy, and low cost detection of SSR markers in non-denaturing polyacrylamide gels. The rapid genotyping of SSR markers can help answer key questions in the genetic research and application field such as genetic diversity analysis and marker assisted selection in molecular breeding programs. The main advantages of this technique is that it's easy to carry out, takes less time and using fewer chemic...