From the previous study by Seo et al. (2015) , we selected 24 polymorphic MS markers for Asian duck discrimination analysis. All MS markers were modified for four types of fluorescence dye (FAM, VIC, NED, and PET) in forward primers. For this analysis, polymerase chain reaction (PCR) amplification was performed in an initial denaturation at 95°C for 10 min followed by 31 cycles of 30 s at 95°C, 30 s at 63°C, 30 s at 72°C, and a final extension at 72°C for 10 min using the C1000 Thermal Cycler (Bio-Rad, USA). A total of 20 μL reaction volume of PCR reagent contained 50 ng of gDNA, 2× Multi HS Prime Taq Premix (GenetBio, Korea), 8 pico mole of each forward and reverse primer, and was adjusted using distilled water. The genotyping mixture contained 1 μL of PCR product, 10 μL of Hi-Di formamide (Applied Biosystems, Waltham, MA, USA), and 0.1 μL of the GeneScan-500LIZ size standard (Applied Biosystems, USA). Fragment analysis was performed using the Genetic Analyzer 3130xl (Applied Biosystems, USA) and the repeat variation results were obtained using Genemapper ver. 4.1 (Applied Biosystems, USA).
Genemapper ver 4
Manufactured by Thermo Fisher Scientific
Sourced in United States
GeneMapper ver. 4.0 is a software application developed by Thermo Fisher Scientific for DNA fragment analysis. It is designed to analyze and interpret genetic data obtained from various laboratory instruments, such as capillary electrophoresis systems. The core function of GeneMapper ver. 4.0 is to process and analyze DNA sequence data, allowing users to identify and quantify specific DNA fragments or alleles.
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Lab products found in correlation
Hi di formamide, by Thermo Fisher Scientific (2 mentions)
Genescan 500 liz size standard, by Thermo Fisher Scientific (1 mentions)
3130xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
C1000 thermal cycler, by Bio-Rad (1 mentions)
Abi prism 3130 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Geneamp pcr system 2700 thermal cycler, by Thermo Fisher Scientific (1 mentions)
3730xl dna analyzer, by Thermo Fisher Scientific (1 mentions)
Mega x software, by Mega Software (1 mentions)
Abi 3130xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
600 liz size standard, by Thermo Fisher Scientific (1 mentions)
Abi 3730xl dna analyzer, by Thermo Fisher Scientific (1 mentions)
5 protocols using genemapper ver 4
1
Polymorphic MS Markers for Asian Duck Discrimination
2
Genetic Diversity Assessment of V. pycnotelma
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In 2012 and 2013, leaf samples from 131 adult individuals were collected from sites same as those from where the herbarium specimens had been collected (Figs. 1c and2). The samples were used to estimate the genetic diversity among the extant populations (Table 2). At all sites, we comprehensively collected samples from each entire patch. Because V. pycnotelma at Site e was locally extinct, we collected leaf samples from a neighbouring population located one kilometre away. The number of individuals was counted at each site.
Genomic DNA was extracted using a modified cetyltrimethylammonium bromide method (Milligan 1992) . The genotypes of each individual, including wild populations and specimens' seedlings, were characterized at nine microsatellite loci. Seven of the nine loci were characterized by Nakahama et al. (2012) : Vpy002, Vpy006, Vpy012, Vpy013, Vpy16, Vpy018 andVpy022 loci were carried out using a GeneAmp PCR System 2700 thermal cycler (Applied Biosystems, Tokyo, Japan) using the following conditions: initial denaturation at 95°C for 15 min, followed by 25 cycles of 30 s at 94°C, 1.5 min at 57°C and 1 min at 72°C, and a final extension for 30 min at 60°C. The PCR product size was measured using an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems) and GeneMapper ver. 4.1 (Applied Biosystems).
Genomic DNA was extracted using a modified cetyltrimethylammonium bromide method (Milligan 1992) . The genotypes of each individual, including wild populations and specimens' seedlings, were characterized at nine microsatellite loci. Seven of the nine loci were characterized by Nakahama et al. (2012) : Vpy002, Vpy006, Vpy012, Vpy013, Vpy16, Vpy018 andVpy022 loci were carried out using a GeneAmp PCR System 2700 thermal cycler (Applied Biosystems, Tokyo, Japan) using the following conditions: initial denaturation at 95°C for 15 min, followed by 25 cycles of 30 s at 94°C, 1.5 min at 57°C and 1 min at 72°C, and a final extension for 30 min at 60°C. The PCR product size was measured using an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems) and GeneMapper ver. 4.1 (Applied Biosystems).
3
Genotypic Analysis of Tobacco Cultivars
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Genotyping data for each Japanese domestic cultivar and modern tobacco varieties K326 (flue-cured variety) and TN90 (burley tobacco) as an outgroup were obtained from 30 simple sequence repeat (SSR) markers and 31 loci. The sequences of SSR markers used are provided in Table 1 . Each forward primer was labeled with a FAM, VIC, TET or NED fluorescent dye (Applied Biosystems) at the 5ʹ end, and a tail sequence (5ʹ-GTGTCTT-3ʹ) was added to each reverse primer. PCR amplification was performed using a QIAGEN Multiplex kit, modified to three set for multiplex reactions in accordance with Komatsu et al. (2020) (link). The PCR products were diluted 50-fold, and 1 μl of Hi-Di formamide (Thermo Fisher Scientific) was added to 10 μl of diluted solution. The mixture was heated for 5 min at 96°C, cooled rapidly on ice, electrophoresed using a 3730xl DNA Analyzer (Life Technology, Applied Biosystems) and analyzed using GeneMapper ver. 4.0 software (Applied Biosystems). A dendrogram was constructed by the neighbor- joining method (Nei et al. 1983 (link)) using Populations 1.2.3 (Langella 1999 ), and a phylogenetic tree was constructed using MEGA X software (megasoftware.net). The reliability of each node was evaluated by 1000 trials of the bootstrap method.
4
Microsatellite Instability Analysis in Cancers
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As previously reported32 (link),33 (link),47 (link), we examined the following five microsatellite loci on chromosomes for MSI based on the revised Bethesda panel52 (link): BAT26, BAT25, D2S123, D5S346, and D17S250. The PCR products were evaluated for MSI by capillary electrophoresis using an ABI 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) and automatic sizing of the alleles using a GeneMapper® Ver. 4.0 (Applied Biosystems). The MSI status was judged according to previous reports40 (link),53 (link),54 (link) (Supplementary Fig. S2 ). In cases that were indistinguishable between MSI and loss of heterozygosity54 (link), the allelic imbalance (AI) ratio was calculated. MSI was determined to be positive when the AI ratio (normal allele 1:normal allele 2/tumor allele 1:tumor allele 2) was <0.67 or >1.35, as previously reported40 (link),53 (link) (Supplementary Fig. S2 ). Lesions were defined as MSI in two or more of the five investigated markers40 (link).
5
Microsatellite Genotyping of Alexandrium ostenfeldii
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