The amplicons of stx1 and stx2 were purified using QIA quick PCR Product extraction kit. (Qiagen Inc., Valencia CA) and then sequenced in Macrogen Company (Korea) using Applied Biosystems 3130 automated DNA Sequencer (USA). The nucleotide sequences were analyzed using BioEdit 7.0.4.1 program. The obtained nucleotide sequences were compared with those available in public domains using NCBI-BLAST server and were deposited in the GenBank Database. Sequence alignments and phylogenetic comparisons of the sequences for the examined genes were performed using MegAlign module of Lasergene DNAStar software.
3130 automated dna sequencer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 3130 Automated DNA Sequencer is a laboratory instrument designed for DNA sequencing. It utilizes capillary electrophoresis technology to determine the precise sequence of nucleotides in DNA samples. The 3130 can process multiple samples simultaneously and provide accurate, high-quality sequencing data.
Automatically generated - may contain errors
Lab products found in correlation
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (2 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Ex taq, by Takara Bio (1 mentions)
Instagene matrix, by Bio-Rad (1 mentions)
Sequencing analysis software, by Thermo Fisher Scientific (1 mentions)
5 protocols using 3130 automated dna sequencer
1
Sequencing and Analysis of stx Genes
2
Molecular Identification of Aeromonas veronii Isolates
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Five well-identified Aeromonas veronii isolates (A (HY1), A (HY2), A (HY3), A (HY4), and A (HY6) were isolated from diseased fish as previously (Youssuf et al. 2020 (link)). The accession number of A. veronii isolates is represented in Table 1 . PCR products were purified using QIA quick PCR Purification kit (QIAGEN, USA) following the manufacturer’s protocol. Then the purified product was sequenced in the forward and/or reverse directions on an Applied Biosystems 3130 automated DNA Sequencer (ABI3130, USA) using a ready reaction Bigdye Terminator V3.1 cycle sequencing kit (Cat. No. 4336817, Perkin- Elmer/Applied Biosystems, Foster City, CA). The resultant sequenced Aeromonas strains were analyzed using the National Center for Biotechnology Information (NCBI), Basic Local Alignment Search Tool (BLAST) program, and the neighbor-joining blast tree against the database of strain types (the most prevalent and pathogenic Aeromonas isolates) and published valid prokaryotic nomenclature (Youssuf et al. 2020 (link)).
Accession numbers of A. veronii isolates
| Samples | NCBI accession number |
|---|---|
| A (HY2) | MK584926 A. veronii HY2 |
| A (HY1) | MK584925 A. veronii HY1 |
| A (HY3) | MK584927 A. veronii HY3 |
| A (HY4) | MK584928 A. veronii HY4 |
| A (HY6) | MK584930 A. veronii HY6 |
3
Automated DNA Sequencing and GenBank Submission
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Sequencing was conducted at Elim Biopharmaceuticals, USA. An extracted conventional PCR product was sequenced in the forward and reverse directions on an Applied Biosystems 3130 Automated DNA Sequencer (ABI 3130 USA)using a ready reaction Big Dye Terminator V3.1 cycle sequencing kit (Perkin-Elmer/Applied Biosystems, Foster City, CA)(Cat. No. 4336817).BLAST® analysis [22 (link)] was used to create sequence characters for GenBank accessions. Sequence results were conducted corresponding to the guides. The results of nucleotide sequencing were submitted to GenBank via Bankit (GenBank n.d.). The sequences were accepted and received accession numbers.
4
HIV-1 Molecular Cloning and Sequencing
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Molecular cloning and determination of the nucleotide sequences of HIV-1 strains passaged in the presence of anti-HIV-1 agents were performed as previously described21 (link). In brief, DNA was extracted from HIV-1-infected MT-4 cells by using the InstaGene Matrix (Bio-Rad, Hercules, CA) and was subjected to cloning, followed by sequence determination. The 1st-round PCR mixture consisted of 1 μl proviral DNA solution, 10 μl Ex-Taq (Takara Bio Inc., Otsu, Japan), and 10 pmol each of the 1st PCR primers in a total volume of 20 μl. The 1st PCR products (1 μl) were used directly in the 2nd-round PCR, and 2nd PCR products were purified with spin-columns (MicroSpin S-400; Amersham Biosciences., Piscataway, NJ), cloned directly using pGEM-T Easy vector (Promega, Fitchburg, WI), and subjected to sequencing with a 3130 automated DNA sequencer (Applied Biosystems, Foster City, CA). The detailed PCR conditions used have been summarized in Supplemental Table 5 .
5
ITS2 Sequencing for Onobrychis Hybrids
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Ripe fruits collected in natural and artificial sympatric populations in 2017 and 2018 from O. normanii and O. chestermanii individuals were harvested and stored at 4 °C until using them for molecular analysis. DNA from pooled seeds from a single fruit was extracted following Doyle and Doyle (1987) . The ITS2 region was amplified by polymerase chain reaction (PCR) using primers annealing with the 3′ region of the 5.8 S and the 5′ region of the 25 S rDNA genes, by using the conditions described in Gögler et al. (2015) and purified by exonuclease I and SAP treatments (Fermentas Inc., Hannover, MD). Purified PCR products were sequenced in both directions using a modification of the Sanger dideoxy method as implemented in a double-stranded DNA cycle sequencing system with fluorescent dyes. Sequence reactions were then run on a 3130 Automated DNA Sequencer and the Sequencing Analysis software (Applied Biosystems, USA) was used to check electropherograms. Sequences were assembled with BioEdit and manually edited. ITS2 sequences showed three indels between O. chestermanii and O. normanii; hybrid fruits were thus recognized by the presence of heterozygosity at these specific sites (Gögler et al. 2015) .
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