Development of a simple and effective silver staining protocol for detection of DNA fragments

Liu, Wenjie; Li, Ronghua; Ayalew, Habtamu; Xia, Yanshi; Bai, Guihua; Yan, Guijun; Siddique, Kadambot H. M.; Guo, Peiguo

doi:10.1002/elps.201700009

Cited by 8 publications

(10 citation statements)

References 10 publications

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“…PCR amplification was performed using an ABI9700 PCR cycler (Applied Biosystems, USA) using the following program: 94°C pre-denaturation for 5 min, 35 cycles of 94°C denaturation for 45 s, varied annealing temperatures ranging from 55–60°C with different primers for 45 s, 72°C extension for 1 min, and a final step at 72°C for 10 min. The PCR products were visualized in a 6% polyacrylamide gel after silver staining [ 18 ].…”

Section: Methodsmentioning

confidence: 99%

Development of EST-SSR markers in flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee) based on de novo transcriptomic assemblies

Chen

Xia

et al. 2017

PLoS ONE

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Flowering Chinese cabbage is one of the most important vegetable crops in southern China. Genetic improvement of various agronomic traits in this crop is underway to meet high market demand in the region, but the progress is hampered by limited number of molecular markers available in this crop. This study aimed to develop EST-SSR markers from transcriptome sequences generated by next-generation sequencing. RNA-seq of eight cabbage samples identified 48,975 unigenes. Of these unigenes, 23,267 were annotated in 56 gene ontology (GO) categories, 6,033 were mapped to 131 KEGG pathways, and 7,825 were assigned to clusters of orthologous groups (COGs). From the unigenes, 8,165 EST-SSR loci were identified and 98.57% of them were 1–3 nucleotide repeats with 14.32%, 41.08% and 43.17% of mono-, di- and tri-nucleotide repeats, respectively. Fifty-eight types of motifs were identified with , , , , and the most abundant. The lengths of repeated nucleotide sequences in all SSR loci ranged from 12 to 60 bp, with most (88.51%) under 20 bp. Among 170 primer pairs were randomly selected from a total of 4,912 SSR primers we designed, 48 yielded unambiguously polymorphic bands with high reproducibility. Cluster analysis using 48 SSRs classified 34 flowering Chinese cabbage cultivars into three groups. A large number of EST-SSR markers identified in this study will facilitate marker-assisted selection in the breeding programs of flowering Chinese cabbage.

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Section: Methodsmentioning

confidence: 99%

Development of EST-SSR markers in flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee) based on de novo transcriptomic assemblies

Chen

Xia

et al. 2017

PLoS ONE

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“…We extracted DNA from 40 leaf samples according to the CTAB method [32]. PCR reactions were performed in the next steps: 5 min of 94 • C pre-denaturation, 45 s of 35 cycles of 94 • C, 45 s of annealing at an optimal temperature ranging from 55 • C to 60 • C, a 45-s extension at 72 • C, then a last 10-min extension at 72 • C. Each of the PCR products were confirmed using 6% polyacrylamide gel electrophoresis [33]. The amount of alleles and Wright's F statistics parameters (Fst), observed heterozygosity (Ho), and expected heterozygosity (He) were quantified by POPGENE 1.32 [34], polymorphism information content (PIC) values were obtained by PIC_CALC software [35].…”

Section: Validation and Application Of Ssr Markersmentioning

confidence: 99%

De Novo Transcriptomic Analysis and Development of EST–SSRs for Styrax japonicus

Zhang

Jiang

et al. 2018

Forests

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Styrax japonicus sieb. et Zucc. is widely distributed in China with ornamental and medicinal values. However, the transcriptome of S. japonicus has not yet been reported. In this study, we carried out the first transcriptome analysis of S. japonicus and developed a set of expressed sequence tag-simple sequence repeats (EST-SSRs). We obtained 338,570,222 clean reads in total, of which the mean GC content was 41.58%. In total, 136,071 unigenes were obtained having an average length of 611 bp and 71,226 unigenes were favorably annotated in the database. In total, we identified 55,977 potential EST-SSRs from 38,611 unigenes, of which there was 1 SSR per 6.73 kb. The di-nucleotide repeats (40.40%) were the most identified SSRs. One set of 60 primer pairs was randomly selected, and the amplified products in S. japonicus were validated; 28 primer pairs successfully produced clear amplicons. A total of 21 (35%) polymorphic genic SSR markers were identified between two populations. In total, 15 alleles were detected and the average number was 6. The average of observed heterozygosity and expected heterozygosity was 0.614 and 0.552, respectively. The polymorphism information content (PIC) value fluctuated between 0.074 and 0.855, with a mean value of 0.504, which was also the middle level. This study provides useful information for diversity studies and resource assessments of S. japonicus.

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“…The technique was initially modified for detection of DNA fragments by Bassam et al 6 in 1991 and then improved by Sanguinetti and coworkers 9 in 1994. The method has been further optimized in the last few decades 6,7,9,10,11,12,13,14,15 . 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 mounting 6 , that limit the application of these protocols 7,11 .…”

Section: Introductionmentioning

confidence: 99%

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

Ling¹,

Deng²,

Li³

et al. 2018

JoVE

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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.

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Development of a simple and effective silver staining protocol for detection of DNA fragments

Cited by 8 publications

References 10 publications

Development of EST-SSR markers in flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee) based on de novo transcriptomic assemblies

Development of EST-SSR markers in flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee) based on de novo transcriptomic assemblies

De Novo Transcriptomic Analysis and Development of EST–SSRs for Styrax japonicus

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

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