The 320-bp fragment of MTHFR containing the 677C>T polymorphism was amplified by polymerase chain reaction (PCR) using the following primers: forward 5'-AAGCAGAGGACTCTCTCTGCCC-3' and reverse 5'-CCCCCAGCCTGTGCGAGGACGGT-3', designed in our laboratory and with an annealing temperature of 57°C for 35 cycles. The fragments obtained were purified using the EZ-10 Spin Column PCR Product Purification kit (Bio Basic, Markham, ON, Canada) following manufacturer instructions and sequenced using an ABI3130 automatic sequencer (Life Technologies, Carlsbad, CA, USA). Sequences were aligned using the BioEdit v7.0.5 software (Hall, 1999) .
Abi3130 automatic sequencer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI3130 automatic sequencer is a laboratory instrument designed for DNA sequencing. It utilizes capillary electrophoresis technology to automate the process of separating and detecting DNA fragments. The core function of the ABI3130 is to perform high-throughput DNA sequence analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ez 10 spin column pcr product purification kit, by Bio Basic (1 mentions)
Methyl primer express software v1, by Thermo Fisher Scientific (1 mentions)
Ez dna methylation lightning kit, by Zymo Research (1 mentions)
Pcr purification kit, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit 50, by Qiagen (1 mentions)
Bigdye terminator cycle sequencing kit v3, by Thermo Fisher Scientific (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
6 protocols using abi3130 automatic sequencer
1
MTHFR 677C>T Polymorphism Genotyping
2
Bisulfite Sequencing for DNA Methylation
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For the methylation analysis, the DNA was subjected to bisulfite modification using EZ DNA Methylation Lightning kit (Zymo Research, Irvine, CA, USA). Primers were designed using Methyl Primer Express software v. 1.0 (Applied Biosystems, Foster City, CA, USA) (Table I). The purified polymerase chain reaction product was sequenced by didesoxyterminal method according to Sanger et al. (13) , using Big Dye Terminator Kit Cycle Sequencing Standard (Life Technologies) version 3.1, followed by capillary electrophoresis in an ABI 3130 automatic sequencer (Life Technologies). The analysis of methylation pattern was assessed in BIQ Analyzer software (14) , and samples with at least 20% of methylated sites were considered hypermethylated (15) .
3
Methylation Analysis of CDH1 Promoter
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For methylation analyses, the samples were subjected to DNA modification using sodium bisulfite
[24 (link)]. A fragment with 22 CpGs of the CDH1 promoter region was amplified using a nested PCR strategy
[25 ]. Fragments obtained were purified using PCR Purification Kit (Invitrogen/Life Technologies, Carlsbad, CA, USA) and sequenced using an ABI3130 automatic sequencer (Applied Biosystems, Foster City, CA, USA). The sequences were aligned with BioEdit v7.0.5
[26 ]. Methylation analyses were run in BiQ Analyzer
[23 (link)] software.
[24 (link)]. A fragment with 22 CpGs of the CDH1 promoter region was amplified using a nested PCR strategy
[25 ]. Fragments obtained were purified using PCR Purification Kit (Invitrogen/Life Technologies, Carlsbad, CA, USA) and sequenced using an ABI3130 automatic sequencer (Applied Biosystems, Foster City, CA, USA). The sequences were aligned with BioEdit v7.0.5
[26 ]. Methylation analyses were run in BiQ Analyzer
[23 (link)] software.
4
Genetic Variation Analysis in Osteogenesis Imperfecta
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Genomic DNA of the probands, their parents and ethnically matched control was extracted from peripheral leukocytes with a QIAamp DNA Mini Kit (50) (Qiagen, Germany). All exons of FKBP10 and PLOD2, exon–intron junctions were amplified by polymerase chain reaction (PCR) in 23 reactions. Primers were designed using the software Oligo 7.0 (Table S1 ). Taq DNA polymerase (Biomed, China) and its standard buffer were used in all reactions under the following conditions: initial denaturation at 95°C for 2 min, followed by 35 cycles at 95°C for 30 s, 53–63°C for 30 s, and 72°C for 45/90 s. Direct sequencing reactions of PCR products were performed using BigDye Terminators Cycle Sequencing Ready Reaction Kit, version 3.1 (Applied Biosystems), and analyzed with an ABI 3130 automatic sequencer (Applied Biosystems) using standard methods. The results of sequencing were compared with the reference nucleotide sequence of FKBP10 (NM_021939.3) and PLOD2 (NM_182943.2). Genetic mutations identified in our patients were submitted to the OI database (https://oi.gene.le.ac.uk ).
5
Genotyping of Groundnut Cultivars
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All samples were used in bulked amplification, using DNA extracted from the plant material. Twenty selected SSR markers were used to genotype 53 groundnut genotypes (Table 2). The SSR sequences were amplified through polymerase chain reaction (PCR). However, due to the poor quality of DNA extracted from 14 genotypes, only 42 genotypes were included in this analysis. PCR products were fluorescently labelled and separated by capillary electrophoresis on ABI 3130 automatic sequencer (Applied Bio systems, Johannesburg, South Africa).
6
Genome Sequencing of Outbreak Strain
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To establish the possible reassortant nature of the outbreak index strain li4129/2014, the complete genome segments A and B were amplified with a OneStep RT-PCR kit in seven and five parts, respectively. The primers are given in Table 1 and the following thermal profile was used: a single cycle of reverse transcription for 30 min at 50°C, 15 min at 95°C for reverse transcriptase inactivation and DNA polymerase activation followed by 40 cycles of 30 s at 95°C, 30 s at 50°C and 30 s at 72°C. If non-specific products were generated, the annealing temperature was increased to 58°C. After agarose gel electrophoresis, the positive bands were cut from the gel and DNAs were extracted with a Qiaquick Gel Extraction Kit (QIAGEN). The sequencing was performed with the primers used in RT-PCR, a BigDye Terminator Cycle sequencing kit v3.1 and an ABI3130 automatic sequencer (Applied Biosystems/ Thermo Fisher Scientific, Waltham, MA). The sequences were edited and the nucleotide identities calculated using the EMBOSS package (Rice et al., 2000) .
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