The total genomic DNA was extracted from the muscle tissues of three specimens of S. niger by the standard phenol–chloroform isoamyl alcohol method [37 ]. The primer pairs, mcb 398 (5′-TACCATGAGGACAAATATCATTCTG-3′) and mcb 869 (5′-CCTCCTAGTTTGTTAGGGATTGATCG-3′) and L2510 (5′-CGCCTGTTTATCAAAAACAT-3′) and H3059 (5′-CCGGTCTGAACTCAGATCACGT-3′), were used to amplify a partial segment of Cytb and 16S rRNA genes [38 (link),39 ]. The 30 mL PCR mix contains 10 pmol of each primer, 20 ng of template DNA, 1X PCR buffer, 1.0–1.5 mM of MgCl2, 0.25 mM of each dNTP, and 1 U of High-fidelity Platinum Taq DNA Polymerase (Invitrogen, Life Science Technologies). The PCR reaction was executed in Veriti Thermal Cycler (Applied Bio systems, Foster City, CA, USA) with the standard thermal profile. The PCR products were cleaned using a QIAquick Gel Extraction Kit (QIAGEN Inc., Germantown, MD, USA) with the usual protocol. The cycle sequencing was performed by using BigDye Terminator ver. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). The bi-directional Sanger sequencing was accomplished by the Genetic Analyzer (Applied Biosystems) housed at Rajiv Gandhi Centre for Biotechnology (RGCB), Thiruvananthapuram, Kerala, India.
Bigdye terminator ver 3.1 cycle sequencing kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The BigDye Terminator v3.1 Cycle Sequencing Kit is a DNA sequencing reagent used in the Sanger sequencing method. The kit provides the necessary reagents, including fluorescently labeled dideoxynucleotides, for performing cycle sequencing reactions to determine the nucleotide sequence of DNA samples.
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Lab products found in correlation
Qiaquick pcr purification kit, by Qiagen (4 mentions)
Veriti thermal cycler, by Thermo Fisher Scientific (3 mentions)
Qiaquick gel extraction kit, by Qiagen (2 mentions)
Bigdye xterminator kit, by Thermo Fisher Scientific (2 mentions)
Abi prism 3100 genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Platinum taq dna polymerase high fidelity, by Thermo Fisher Scientific (2 mentions)
Applied biosystems 3500xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Abi prism 3130xl sequencer, by Thermo Fisher Scientific (1 mentions)
Pcr purification kit, by Roche (1 mentions)
Abi prism 3130xl, by Thermo Fisher Scientific (1 mentions)
Primestar gxl polymerase, by Takara Bio (1 mentions)
T vector pmd20, by Takara Bio (1 mentions)
Primescript reverse transcriptase, by Takara Bio (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
Qiaquickr gel extraction kit, by Qiagen (1 mentions)
Abi 3730 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Applied biosystems 3500 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Isogen ls, by Nippon Gene (1 mentions)
11 protocols using bigdye terminator ver 3.1 cycle sequencing kit
1
Cytb and 16S rRNA Genes Amplification
2
Sequencing and Analysis of NNV Genotypes
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The obtained PCR product (605 bp) was sequenced for four NNV-positive specimens from different organs using the QIAquick PCR Purification Kit (QIAGEN, Valencia, CA, USA) for PCR product purification. A BigDye Terminator ver. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) was used for the sequence reaction of the purified PCR products. Sanger sequencing was conducted at Macrogen, Korea, using an ABI PRISM 3730XL analyzer (96 capillary types). Nucleotide sequencing was performed by using the primer sets described above. The bio-edit package ver. 7.2 software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html ) was used to analyze the obtained sequence data compared to other sequences retrieved from GenBank by a BLAST homology search (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html ). The nucleotide alignments of the selected sample sequences were compared with those of other NNV genotype sequences.
3
Molecular Characterization of Rotavirus
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To further investigate RVA genotypes in G and P typing and sequence RVA genomes, all VP7 and VP4 PCR-positive samples were sequenced. The sequences were determined directly from the PCR products with a BigDye terminator ver. 3.1 cycle sequencing kit using an Applied Biosystems 3500XL Genetic Analyzer (Applied Biosystems, Waltham, MA, United States). Phylogenetic analyses were performed based on the nucleotide sequences of the amplified VP7 and VP4 genes by RT-PCR. Representative strains for each G- and P-genotype successfully sequenced were selected for phylogenetic analyses. Reference sequences were retrieved from the DNA Data Bank of Japan/European Molecular Biology Laboratory/GenBank databases. Alignments were performed using the CLUSTAL X (version 1.83) software, and phylogenetic trees were constructed by the neighbor-joining method. To confirm the reliability of phylogenetic tree analysis, bootstrap resampling, and reconstruction were carried out 1,000 times. These analyses were carried out using Molecular Evolutionary Genetic Analysis (MEGA4) software (Tamura et al., 2007 (link)). The gene sequences described in the present study have been deposited in the GenBank database under accession numbers LC597263–LC597321 and MW840090–MW840135.
4
Mitochondrial COI Sequencing of Pigeons
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Mitochondrial COI sequence was analyzed for one sample of the 10 Egyptian breeds and of Japanese feral pigeons. Moreover, one sample was sequenced from the four wild pigeon species: Emerald dove (Chalcophaps indica), Oriental turtle dove (Streptopelia orientalis), Whistling green pigeon (Treron formosae), and white-bellied green pigeon (Treron sieboldii) for comparison. The primers and polymerase chain reaction (PCR) condition are the same as described by Ramadan et al. [10 ].
The amplified products were purified using PCR Purification Kit (Roche, Mannheim, Germany), and the resultant products were sequenced using the same primers and the Big Dye Terminator ver. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) according to the standard protocol and electrophoresed on an ABI PRISM 3130xl sequencer (Applied Biosystems). The MEGA 6 Software (https://www.megasoftware.net ) [16 (link)] was used for sequences alignment and to infer the phylogenetic relationships based on neighbor-joining [17 (link)] methods [18 (link)].
The amplified products were purified using PCR Purification Kit (Roche, Mannheim, Germany), and the resultant products were sequenced using the same primers and the Big Dye Terminator ver. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) according to the standard protocol and electrophoresed on an ABI PRISM 3130xl sequencer (Applied Biosystems). The MEGA 6 Software (
5
Mitochondrial DNA Barcoding of Shrew Species
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The genomic DNA was extracted from both the holotype and the paratype specimen by the standard phenol–chloroform isoamyl alcohol method55 . The extracted DNA was visualized through 1% agarose gel electrophoresis. The published primer pair (mcb 398: 5′-TACCATGAGGACAAATATCATTCTG-3′ and mcb 869: 5′-CCTCCTAGTTTGTTAGGGATTGATCG-3′)56 (link) was used to amplify the widely applied mitochondrial Cytochrome b (mtCytb) gene segment for the identification of shrew species7 ,57 . The 25 ml PCR mixture comprises 10 pmol of each primer, 20 ng of DNA template, 1X PCR buffer, 1.0–1.5 mM of MgCl2, 0.25 mM of each dNTPs, and 1 U of Platinum Taq DNA Polymerase High fidelity (Invitrogen). The PCR reaction was performed in Veriti Thermal Cycler (Applied Biosystems) with the published thermal profile. The PCR products were purified using a QIAquick Gel Extraction Kit (QIAGEN) with standard protocol. The cycle sequencing was executed by using BigDye Terminator ver. 3.1 Cycle Sequencing Kit (Applied Biosystems) and 3.2 pmol of each primer on Veriti Thermal Cycler. The products were cleaned by BigDye X-terminator kit (Applied Biosystems) with standard protocol and subsequently bidirectional sequenced by the 48 capillary 3730 Genetic Analyzer (Applied Biosystems).
6
Multiplex PCR Sequencing of SCN5A
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The coding region (exon 2-exon 28) of SCN5A was amplified by multiplex polymerase chain reaction (PCR) using newly designed primers (Table 1 ). PCR conditions were as follows: after an initial denaturation at 95℃ for 15 min, denaturation at 94℃ for 30 sec, annealing at 68-70℃ for 30-60 sec, and extension at 72℃ for 60-90 sec were repeated for 30-35 cycles. Following multiplex PCR, the reaction mixture was electrophoresed in a 2% agarose gel and stained with ethidium bromide (EtBr). Amplified PCR products were purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany), and directly sequenced using the BigDye Terminator Ver 3.1 cycle sequencing kit (Applied Biosystems, Foster city, CA, USA) and an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Sequencing results were compared with reference sequences (SCN5A/NM_198056.2/ENSG00000183873/ENST00000333535) using the alignment program BLAST 2.0 of the national center of biotechnology information (NCBI; Bethesda, MD, USA), and the portion of variation that occurred was determined.
7
RABV Identification via RT-PCR and Sequencing
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The RT-PCR products were purified with a QIAquick PCR Purification Kit (Qiagen, Germany). The purified products were sequenced directly using the same primer applied in RT-PCR. A cycle sequencing reaction was conducted with the BigDye Terminator ver. 3.1 Cycle Sequencing Kit (Applied Biosystems, USA). The sequencing products were obtained with the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) at the Eijkman Institute, Jakarta. The sequencing results were aligned using ClustalW in the Mega4 software [35 (link)] and RABV was identified by BLAST analysis [1 (link)]. The N gene target sequence was 609 bp, corresponding to the nucleotides at positions -22 to 587. All nucleotide sequences have been submitted to GenBank and assigned accession numbers (Table 1 ).
8
Isolation and Sequencing of Carnation Chalcone Isomerase Genes
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Genomic DNAs and total RNAs were isolated from ‘4-94-1 MR’ and ‘120 MOR’, ‘129 MOR’ and ‘129 MOR-MOR1’ petals at developmental stage 3 using a DNeasy Plant Mini Kit and a RNeasy Plant Mini Kit (Qiagen), respectively. First-strand cDNA was synthesized from 1 μg total RNA using oligo(dT)s and PrimeScript reverse transcriptase (Takara Bio). CHI (Dca60978 and Dca60979) cDNA fragments were obtained using specific primer sets (Dca60978 Fwd and Dca60978 Rv for Dca60978, Dca60979 Fwd and Dca60979 Rv for Dca60979) based on the nucleotide sequences of the cultivar ‘Francesco’ in the database Carnation DB (Yagi et al. 2014 (link)) (Supplemental Table 1 ). PCR was performed using Prime Star GXL polymerase (Takara Bio) under the following conditions: 2 min denaturation at 94°C, then 35 cycles of 10 s denaturation at 98°C, 15 s annealing at 55°C, 20 s extension at 68°C, and a final 1 min extension at 68°C. After an adenylation reaction, the amplified DNAs were introduced into the T-vector pMD20 (Takara Bio). The sequences of the genomic and cDNA fragments were determined using a BigDye terminator ver.3.1 cycle sequencing kit and an ABI PRISM 3130xl (Applied Biosystems). Nucleotide structures of Dca60978 and Dca60979, without the transposable element dTdic1, were confirmed by genomic and cDNA sequencing. Dca60979 with the dTdic1 insert was identified by its genomic sequence.
9
Mitochondrial Cytochrome b Sequencing for Shrew Identification
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The genomic DNA was extracted from both holotype and paratype specimens by standard phenolchloroform isoamyl alcohol method 54 . The extracted DNA was visualized through 1% agarose gel electrophoresis. The published primer pair (mcb 398: 5′-TACCATGAGGACAAATATCATTCTG-3′ and mcb 869: 5′-CCTCCTAGTTTGTTAGGGATTGATCG-3′) 55 was used to amplify the widely applied mitochondrial Cytochrome b (mtCytb) gene segment for the identi cation of shrew species 56, 57 . The 25 ml PCR mixture comprises 10 pmol of each primer, 20 ng of DNA template, 1X PCR buffer, 1.0-1.5mM of MgCl2, 0.25mM of each dNTPs, and 1 U of Platinum Taq DNA Polymerase High delity (Invitrogen, Life Science Technologies). The PCR reaction was performed in Veriti®Thermal Cycler (Applied Biosystems, Foster City, CA) with the published thermal pro le. The PCR products were purified using a QIAquickR Gel extraction kit (Qiagen Inc., Germantown, MD) with standard protocol. The cycle sequencing was executed by using BigDye®Terminator ver. 3.1 Cycle Sequencing Kit (Applied Biosystems, Inc.) and 3.2 picomoles of each primer on Veriti®Thermal Cycler. The products were cleaned by BigDye X-terminator kit (Applied BiosystemsInc.) with standard protocol and subsequently bidirectional sequenced by the 48 capillary ABI 3730 Genetic Analyzer.
10
Sequencing of Influenza Virus Genes
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Total RNA was extracted from allantoic fluids using ISOGEN-LS (NIPPON GENE CO., LTD., Tokyo, Japan). For nucleotide sequencing of AIV genes, the viral RNA was transcribed into cDNA using the universal 12-mer primer (5 0 -AGC RAA AGC AGG-3 0 ) and Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) at 42 °C for 60 min, followed by 70 °C for 10 min. Using the cDNA as a template, PCR was conducted to amplify the entire length of all eight viral segments using segmentspecific primers as described by Hoffmann et al. [32] and Obenauer et al. [33] . The resulting PCR products were separated by 1 % agarose gel electrophoresis and purified using either a QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) or a Gene Clean II Kit (Biogene, Inc., USA). The purified PCR products were used as templates for sequencing reactions using a BigDye Terminator ver.
3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). Sanger sequencing was performed using an Applied Biosystems 3500 Genetic Analyzer (Life Technologies, Carlsbad, CA). The primer sets described above as well as additional overlapping primers that were designed as needed (sequences available upon request) were used for nucleotide sequencing.
3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). Sanger sequencing was performed using an Applied Biosystems 3500 Genetic Analyzer (Life Technologies, Carlsbad, CA). The primer sets described above as well as additional overlapping primers that were designed as needed (sequences available upon request) were used for nucleotide sequencing.
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