Name: a fast silver staining protocol enabling simple and efficient detection of ssr markers using a non-denaturing polyacrylamide gel
Uploaded: 2018-04-20T13:30:00
Description: 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

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>
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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 ...

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