Genotyping was performed with the Invader assay (Third Wave Technologies, Madison, WI, USA) and Taq Man Allelic discrimination assay. Reagents were purchased from Applied Biosystems (Foster City, CA, USA). Taq Man probes that can distinguish SNPs after polymerase chain reaction (PCR) were designed and synthesized by Applied Biosystems. One allelic probe was labelled with the fluorescent FAM dye, and the other with the fluorescent VIC dye. PCR was performed with the Taq Man Universal Master Mix with primers at concentrations of 225 nM and Taq Man MGB probes at concentrations of 50 nM. Reactions were performed in 382 well plates in a total volume of 3 μL using 3.0 ng genomic DNA. The plates were then placed in a GeneAmp PCR system 9700 (Applied Biosystems) and heated at 95°C for 10 min, followed by 40 cycles at 92°C for 15 sec and at 60°C for 1 min, with a final incubation at 25°C. The fluorescent intensities of each well in the plates were then read by the Prism 7900HT instrument (Applied Biosystems). Fluorescent data files from each plate were analyzed with the SDS 2.0 allele calling software (Applied Biosystems). Several data points were eliminated to preserve the reliability of the assay system (missing data due to poor signal intensity < 1.1%) [12 (link)].
Sds 2.0 allele calling software
Manufactured by Thermo Fisher Scientific
SDS 2.0 allele calling software is a bioinformatics application designed for analyzing genetic data. It is used to identify and report alleles, which are different versions of a specific gene, from DNA sequence data.
Automatically generated - may contain errors
2 protocols using sds 2.0 allele calling software
1
Genotyping SNPs Using Taq Man Assay
2
Genotyping SNPs using Invader and TaqMan Assays
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Genotyping was performed using the Invader assay (Third Wave Technologies, Madison, WI, USA) and TaqMan allelic discrimination assay. Reagents were purchased from Applied Biosystems (Foster City, CA, USA). TaqMan probes that can distinguish SNPs after a polymerase chain reaction (PCR) were designed and synthesized by Applied Biosystems. One allelic probe was labeled with the fluorescent FAM dye and the other was labeled with the fluorescent VIC dye.
PCRs were conducted using the TaqMan Universal Master Mix with primers at a concentration of 225 nM and TaqMan MGB probes at a concentration of 50 nM. The reactions were conducted in 382-well plates in a total volume of 3 μL using 3.0 ng genomic DNA. The plates were then placed in a GeneAmp PCR system 9700 (Applied Biosystems) and heated at 95℃ for 10 min, followed by 40 cycles at 92℃ for 15 s and at 60℃ for 1 min, with a final incubation at 25℃. The fluorescent intensities of each well in the plates were read using Prism 7900HT (Applied Biosystems). Fluorescent data files from each plate were analyzed using the SDS 2.0 allele calling software (Applied Biosystems). Several data points were eliminated to preserve the reliability of the assay system (missing data due to poor signal intensity <1.1%).12 (link),14 (link)
PCRs were conducted using the TaqMan Universal Master Mix with primers at a concentration of 225 nM and TaqMan MGB probes at a concentration of 50 nM. The reactions were conducted in 382-well plates in a total volume of 3 μL using 3.0 ng genomic DNA. The plates were then placed in a GeneAmp PCR system 9700 (Applied Biosystems) and heated at 95℃ for 10 min, followed by 40 cycles at 92℃ for 15 s and at 60℃ for 1 min, with a final incubation at 25℃. The fluorescent intensities of each well in the plates were read using Prism 7900HT (Applied Biosystems). Fluorescent data files from each plate were analyzed using the SDS 2.0 allele calling software (Applied Biosystems). Several data points were eliminated to preserve the reliability of the assay system (missing data due to poor signal intensity <1.1%).12 (link),14 (link)
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