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

Summary

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.

Abstract

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.

Introduction

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 traits^3,

Protocol

1. Preparation of PCR Products of SSR Markers

1. Prepare all chemicals and reagents for PCR reactions including template DNA (30 ng/µL), 2× PCR master mix (containing 2× PCR buffer, 0.4 mM of each dNTP, 3 mM of MgCl₂, 0.1 U/µL of Taq DNA polymerase and dyes), 10 µM 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 '-GCACCTTTGGTTATCCGACA-3 ' and reverse primer sequence: 5 '-TGCTTTAAGTCATGTACCAAATTGA-3 ') and PT50903 (forward primer sequence: 5 '-AAATTTCTTTCGGTGTGATAACTG-3&....

Results

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.......

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PCR master mix (Green Taq Mix)	Vazyme Biotech Co. Ltd, China	#P131-03
50-2000 bp DNA Ladder	Bio-Rad, USA	#170-8200
DL500 DNA marker	Takara Bio Inc., Japan	#3590A
Tris base	Sangon Biotech Shanghai, China	#77-86-1
Boric acid	Sangon Biotech Shanghai, China	#10043-35-3
EDTA-Na2	Guangzhou Chemical Reagent Factory, China	#6381-92-6
Acrylamide	Sangon Biotech Shanghai, China	#79-06-1
N,N'-methylene-bis-acrylamide	Sangon Biotech Shanghai, China	#110-26-9
N,N,N',N'-Tetramethylethylenediamine	Sangon Biotech Shanghai, China	#110-18-9
Ammonium persulfate	Guangzhou Chemical Reagent Factory, China	#7727-54-0
Bind-silane	Solarbio Beijing, China	#B8150
AgNO3	Sinopharm Chemical Reagent Beijing Co.,Ltd, China	#7761-88-8
Formaldehyde	Tianjin DaMao Chemical Reagent Factory, China	#50-00-0
NaOH	Guangzhou Chemical Reagent Factory, China	#1310-73-2
Acetic acid	Guangzhou Chemical Reagent Factory, China	#64-19-7
Na2CO3	Tianjin DaMao Chemical Reagent Factory, China	#497-19-8
Ethanol	Guangzhou Chemical Reagent Factory, China	#64-17-5
HNO3	Guangzhou Chemical Reagent Factory, China	#7697-37-2
Na2S2O3.5H2O	Sinopharm Chemical Reagent Beijing Co.,Ltd, China	#10102-17-7
Eriochrome black T(EBT)	Tianjin DaMao Chemical Reagent Factory, China	#1787-61-7
Plastic tray	Shanghai Yi Chen Plastic Co., Ltd, China	-
TS-1 Shaker	Qilinbeter JiangSu, China	-
BenQ M800 Scanner	BenQ, China	-
DYY-6C Power supply	Beijing Liuyi Instrument Factory, China	-
High throughout vertical gel systems, JY-SCZF	Beijing Tunyi Electrophoresis Co., Ltd, China	-