SNP genotyping was conducted using the MassARRAY system as described by Kaga et al. [7 (link)]. Briefly, multiplex PCR with a primer set 1st_PCRP and 2nd_PCRP followed by a template-directed single base extension with a primer UEP_SEQ at the SNP site (rj2 cDNA sequence position 1470 of soybean cultivar Williams 82) were conducted using the iPLEX Gold kit (Agena Bioscience, Inc. San Diego, CA, USA) following the manufacture’s protocol. The reaction mixture was dispensed onto a silicon matrix preloaded SpectroCHIP (Agena Bioscience, Inc.) using Nanodispenser (Agena Bioscience, Inc.) and analyzed by Compact MassARRAY MALDI-TOF (Agena Bioscience, Inc.). The genotypes were determined using MassARRAY Typer4.0 (Agena Bioscience, Inc.).
Iplex gold kit
Manufactured by Agena
Sourced in United States, Germany
The IPLEX Gold Kit is a compact and portable industrial videoscope designed for visual inspection and documentation. It features a high-resolution camera and interchangeable probes for examining hard-to-reach areas. The kit includes the IPLEX Gold videoscope, various probe options, and accessories to support a range of industrial inspection tasks.
Automatically generated - may contain errors
4 protocols using iplex gold kit
1
SNP Genotyping by MassARRAY System
2
Multiplex Genotyping of FH Mutations
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The FH mutations to be studied were selected according to the known mutation frequencies from our previous study
9)
. Sequences covering the selected alterations were taken from the databases of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) and Ensembl (http://www.ensembl.org/ index.html).
The entire multiplex reaction process was performed according to the manufacturer’s instructions, including PCR amplification, shrimp alkaline phosphatase treatment, and primer extension reaction using the iPLEX Gold assay (Agena Bioscience, San Diego, California), which have been described in detail before
9)
. Briefly, genomic DNA was amplified via multiplex PCR (PCR Accessory and Enzyme Kit; Agena Bioscience) and unincorporated dNTPs were deactivated. The single-base extension reaction, consisting of the iPLEX enzyme, terminator mix, and extension primer mix, were subject to thermocycling conditions (iPLEX Gold Kit; Agena Bioscience). After desalting, the PCR products were spotted on SpectroCHIP II arrays using a MassARRAY nanodispenser and analyzed using the MassARRAY platform. Mass signals for the different alleles were captured with high accuracy by MALDI-TOF MS, and Typer v4.0 (Agena Bioscience) was used to process the raw data obtained from the assays.
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The FH mutations to be studied were selected according to the known mutation frequencies from our previous study
The entire multiplex reaction process was performed according to the manufacturer’s instructions, including PCR amplification, shrimp alkaline phosphatase treatment, and primer extension reaction using the iPLEX Gold assay (Agena Bioscience, San Diego, California), which have been described in detail before
3
Genotyping Assays via IPLEX MassARRAY
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Genotyping was performed via IPLEX MassARRAY PCR using the Agena platform (Agena Bioscience, San Diego, CA, USA). IPLEX MassARRAY PCR and extension primers were designed from sequences containing each target SNP and 150 upstream and downstream bases with Assay Designer 4.0.0.2 (Agena Bioscience, San Diego, CA, USA) using the default settings. Single base extension reactions were performed on the PCR reactions with the iPLEX Gold Kit (Agena Bioscience) and 0.8 µL of the custom UEP pool. PCR reactions were dispensed onto SpectroChipArrays with a Nanodispenser (Agena Bioscience). An Agena Bioscience Compact MassArray Spectrometer was used to perform MALDI–TOF mass spectrometry according to the iPLEX Gold Application Guide. The Typer 4 software package (Agena Bioscience) was used to analyse the resulting spectra, and the composition of the target bases was determined from the mass of each extended oligo. These panels were designed in collaboration with PATIA, and genotyping was performed using the Agena platform located at the Epigenetics and Genotyping laboratory, Central Unit for Research in Medicine (UCIM), Faculty of Medicine, University of Valencia, Valencia, Spain.
4
Multiplex SNP Genotyping by iPLEX Assay
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A total of 111 AIM-SNPs distributed in four multiplexes (ME1-ME4) were amplified by UCPH and USC using the iPLEX ® Gold Kit (Agena Bioscience GmbH, Hamburg, Germany) in a final reaction volume of 5 μL. Information about the markers and primers included in each iPLEX multiplex is outlined in Supplementary Table S2.
The PCR amplification mix consisted of 1 µL DNA, 0.5 µL 10x Buffer, 0. The PCR products were treated with Shrimp Alkaline Phosphatase (SAP) to dephosphorylate the remaining dNTPs from the PCR. Each reaction contained 0.17 µL SAP buffer, 0.30 µL SAP enzyme and 1.53 µL of water. The SAP reaction was carried out at 37ºC for 40 min and 85ºC for 5 min.
The SBE reaction was carried out with 7 μL SAP-treated PCR products and 2 μl iPLEX ® mix (Agena Bioscience). The iPLEX ® mix contained 0.2 µL 10x iPLEX ® buffer, 0.2 µL iPLEX ® -Termination mix, 0.94 µL primer mix (DNA Technology, Denmark), 0.04 µL iPLEX ® -enzyme and 0.62 µL water. The SBE reaction was carried out with the following conditions: denaturation for 30s at 94˚C followed by 40 cycles consisting of 3 steps: 5s at 94˚C, 5s at 52˚C and 5s at 80˚C, where steps 2 and 3 were repeated 5 times in each cycle. The final extension consisted of 3 min at 72˚C. Samples were analyzed with the MassARRAY ® System (Agena Bioscience) using the autorun settings. All samples were run in duplicate.
The PCR amplification mix consisted of 1 µL DNA, 0.5 µL 10x Buffer, 0. The PCR products were treated with Shrimp Alkaline Phosphatase (SAP) to dephosphorylate the remaining dNTPs from the PCR. Each reaction contained 0.17 µL SAP buffer, 0.30 µL SAP enzyme and 1.53 µL of water. The SAP reaction was carried out at 37ºC for 40 min and 85ºC for 5 min.
The SBE reaction was carried out with 7 μL SAP-treated PCR products and 2 μl iPLEX ® mix (Agena Bioscience). The iPLEX ® mix contained 0.2 µL 10x iPLEX ® buffer, 0.2 µL iPLEX ® -Termination mix, 0.94 µL primer mix (DNA Technology, Denmark), 0.04 µL iPLEX ® -enzyme and 0.62 µL water. The SBE reaction was carried out with the following conditions: denaturation for 30s at 94˚C followed by 40 cycles consisting of 3 steps: 5s at 94˚C, 5s at 52˚C and 5s at 80˚C, where steps 2 and 3 were repeated 5 times in each cycle. The final extension consisted of 3 min at 72˚C. Samples were analyzed with the MassARRAY ® System (Agena Bioscience) using the autorun settings. All samples were run in duplicate.
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