Total DNA was extraction from cell pellets using TIANamp Genomic DNA Kit (TIANGEN). Then the genomic fragments, including PRDM15, ZBTB21, and MX1-ABCG1 were amplified using 2xTaq Master Mix (vazyme Bio-tech) with primers in table S2 . PCR productions were purified and sequenced in both direction with BigDye Terminator Cycle Sequencing Kit on an ABI PRISM 3730 DNA analyser (Applied Biosystems). Sequences of each segment were proofread and assembled with DNASTAR 5.0 (DNASTAR Inc., Madison, WI, USA).
Abi prism 3730 dna analyser
Manufactured by Thermo Fisher Scientific
Sourced in Belgium
The ABI Prism 3730 DNA Analyser is a capillary electrophoresis system designed for DNA sequencing and fragment analysis. It utilizes fluorescence detection technology to analyze and separate DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Bigdye terminator v3.0 kit, by Thermo Fisher Scientific (2 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Taq master mix, by Vazyme (1 mentions)
Tianamp genomic dna kit, by Tiangen Biotech (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Qiasymphony sp instrument, by Qiagen (1 mentions)
Onestep rt pcr kit, by Qiagen (1 mentions)
4 protocols using abi prism 3730 dna analyser
1
Targeted DNA Sequencing of Key Genes
2
Phylogenetic Analysis of Porcine Reproductive and Respiratory Syndrome Virus 1
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PRV1-positive samples were subsequently characterised by Sanger sequencing using RT-PCR to amplify the entire F gene [15 (link)]. The PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). Sequencing reactions were performed with a BigDye Terminator v3.0 kit (Applied Biosystems, Lennik, Belgium) and analysed with an ABI Prism 3730 DNA Analyser (Applied Biosystems). Phylogenetic analysis was performed by comparing the obtained sequences with other PRV1 sequences available in GenBank and aligning them using ClustalW. A phylogenetic tree was constructed using the Maximum Likelihood method and the General Time Reversible model (G + I), identified using ModelFinder selection, with bootstrap analysis (1000 replicates) using MEGA10 software. The HRV1 F-gene sequence (GenBank accession: NC003461) was used as an outgroup.
Nucleotide homology percentages were determined via NCBI BLASTn analysis.
Nucleotide homology percentages were determined via NCBI BLASTn analysis.
3
Cryptosporidium Sequencing and Analysis
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All secondary PCR products were sequenced using the same secondary PCR primers on an ABI PRISM™ 3730 DNA Analyser (Applied Biosystems, Carlsbad, CA, USA) using a BigDye Terminator v3·1 Cycle Sequencing kit (Applied Biosystems). The accuracy of the sequencing data were confirmed by sequencing the PCR products in both directions. Further PCR products from specific DNA preparations were sequenced as required. Nucleotide sequences obtained in the present study were subjected to BLAST searches (http://www.ncbi.nlm.nih.gov/blast/ ) and then analyzed and aligned with each other and the published reference sequences of Cryptosporidium in GenBank, using ClustalX 1.81 (http://www.clustal.org/ ).
4
Bovine Rotavirus Genotyping Protocol
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Infected MDBK cells were scraped off the plates and homogenised using three cycles of freezing and thawing. Viral RNA was extracted from 250 μL of supernatant using the QIAsymphony™ SP Instrument (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Oligonucleotide primers for BRV3 detection and identification described by Maidana et al. (2012) [24 (link)] were used to amplify a 328 bp segment of the consensus BRV3 Matrix (M) gene by RT-PCR using the commercial Qiagen One-Step RT-PCR kit (Qiagen). The PCR products were purified using the Qiaquick PCR Purification Kit (Qiagen). Sequencing reactions were performed with BigDye Terminator v3.0 kit (Applied Biosystems, Lennik, Belgium) and analysed with an ABI Prism 3730 DNA Analyser (Applied Biosystems).
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