PCR products were purified using the PCR DNA Purification Kit (Applied Biosystems, California, USA) and sequenced using 10 ng of purified DNA and 3.2 pmoles of each primer set in a final volume of 20 μL. After amplification in the same conditions as the PCR step (Section 2.3.3 ), products were purified according to the BigDye Terminator Purification X Kit protocol (Applied Biosystems, California, USA) and sequenced on a 3130 sequencer Genetic Analyzer (Applied Biosystems, California, USA). The sequences were compared to Staphylococcus aureus and Staphylococcus pasteuri gene sequences available at the GenBank database (http://www.ncbi.nlm.nih.gov/Genbank/index.html ). Multiple sequence alignments were performed using Clustal W (Kyoto University, Bioinformatics Center; http://www.genome.jp/tools/clustalw/ ).
Sequencer 3130 genetic analyzer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Sequencer 3130 Genetic Analyzer is a capillary electrophoresis instrument designed for DNA sequencing applications. It utilizes four-color fluorescence detection and automated sample loading to provide high-resolution genetic analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pcr dna purification kit, by Thermo Fisher Scientific (3 mentions)
Bigdye terminator purification x kit, by Thermo Fisher Scientific (3 mentions)
Transcriptor one step rt pcr kit, by Roche (2 mentions)
Qiaamp viral rna mini kit, by Qiagen (2 mentions)
Platinum taq polymerase enzyme, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Gfx pcr dna and gel band purification kit, by GE Healthcare (1 mentions)
Veriti thermal cycler, by Thermo Fisher Scientific (1 mentions)
Sequencher program, by Gene Codes (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Onestep rt pcr kit, by Qiagen (1 mentions)
9 protocols using sequencer 3130 genetic analyzer
1
Staphylococcus Species Identification via DNA Sequencing
2
Identification of Staphylococcus in Salami Samples
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Amplification of the V5 region of 16S rDNA fragment was performed using 50 ng of DNA templates from the 65 strains found in the salami samples. PCR was performed under the following conditions: 95°C for 10 min, followed by 30 cycles at 95°C for 30 s, 60°C for 30 s and 72°C for 45 s, and a final extension at 72°C for 10 min. PCR products were purified using the PCR DNA Purification Kit (Applied Biosystems, California, USA) and sequenced using 20 ng purified DNA and 13 μL of primer sets in a final volume of 20 μL. After amplification, products were purified according to the protocol of the BigDye Terminator Purification X Kit (Applied Biosystems, California, USA) and sequenced on a 3130 sequencer Genetic Analyzer (Applied Biosystems, California, USA). The sequences were compared to the 16S rDNA gene sequences of Staphylococcus species available at the GenBank database (http://www.ncbi.nlm.nih.gov/Genbank/index.html ). Multiple sequence alignments were performed using Clustal W (Kyoto University, Bioinformatics Center; http://www.genome.jp/tools/clustalw/ ).
3
Identification of Staphylococcus in Minas Frescal Cheese
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Amplification of the V5 region of 16S rDNA was performed using 50 ng of DNA templates from the 60 strains isolated from Minas Frescal cheeses. A PCR was performed under the following conditions: 95°C for 10 min, followed by 30 cycles at 95°C for 30 s, 60°C for 30 s and 72°C for 45 s, and a final extension at 72°C for 10 min. The PCR products were purified using the PCR DNA Purification kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA) and sequenced using 20 ng of purified DNA and 13 μL of primer set of forward and reverse 16S rDNA (Table 1) in a final volume of 20 μL. After the amplification, products were purified according to the protocol of the BigDye Terminator Purification X kit (Applied Biosystems) and sequenced in a 3130 sequencer Genetic Analyzer (Applied Biosystems). Staphylococcus species identification was performed by comparing the partial sequences of 16S rDNA to the sequences available at GenBank database (http://www.ncbi.nlm.nih.gov/Genbank/index.html). The nucleotide sequences were then submitted to Gen-Bank, which provided new accession number to each isolated strains of Staphylococcus species available in the GenBank database. Multiple sequence alignments were performed using ClustalW (Kyoto University, Bioinformatics Center; http://www.genome.jp/tools/ clustalw/).
4
Fungal Identification by Morphology and DNA
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The fungi that developed were quantified and grouped into morphotypes and identified using conventional taxonomic keys [18 , 19 ]. The morphological identification was associated with DNA sequencing analysis. Identification by DNA sequencing initiated with culture DNA was extracted with the kit QIAamp Tissue and Blood (Qiagen, Hilden, Germany) according to manufacturer. The PCR was performed in accordance with [20 ] from the amplification of the regions ITS (ITS1-5′ TCC GTA GGT GAA CCT GCGG 3′ and ITS4-5′ TCC TCC GCT TAT TGA TAT GC 3′). The following were used: 40 ng of DNA in a final volume of 30 µL, with 1X reaction buffer (200 mM TrisHCl (pH 8.4), 500 mM KCl); 0.2 mM of each nucleotide; 2 mM of magnesium chloride (MgCl2); 50 ng of each primer; and 2.5U of Platinum® Taq polymerase enzyme (Invitrogen, Brazil). The amplicons were sequenced according to the instructions from the manufacturer of the kit “BigDye®Terminator v3.1 Cycle Sequencing Kit” (Applied Biosystems) and capillary electrophoresis was performed in an ABI 3130 Genetic Analyzer sequencer. The sequences were analyzed in the Sequencher 5.4.6 program and then compared with existing sequences in the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi ) and the Wester Dijk Fungal Biodiversity Institute (https://wi.knaw.nl/page/Pairwisealignment ).
5
Genotype Validation via Sanger Sequencing
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The imputed genotype information for each family member for two genetic variants located in the DPY19L2 locus, was confirmed by direct automated sequencing. DPY19L2 exons 3 and 8 were amplified using previously described PCR primers (Chianese et al. , 2015) . Sequences analyses were carried out on a 3130 Genetic Analyzer sequencer (Applied Biosystems, Hitachi, Japan).
6
Recombinant NDV_HKur Characterization
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RNA was isolated from allantoic fluid after the first and second passage in ECE of recombinant NDV_HKur using the QIAamp® Viral RNA Mini Kit (Qiagen). Genomic regions, encoding the proteolytic cleavage site within the NDV F gene, as well as the region, encoding the inserted PPRV H, were transcribed into cDNA and amplified, using the Transcriptor One-Step RT PCR Kit (Roche Applied Science, Mannheim, Germany). Sanger-sequencing (Sequencer 3130 Genetic Analyzer, Applied Biosystems, Foster City, CA, USA) was used to confirm virus identity. Virus stock was prepared from allantoic fluid of the second ECE passage.
7
Verification of Recombinant NDV_GRABV Identity
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RNA was isolated from allantoic fluid after the first, second and 10th passage in ECE of recombinant NDV_GRABV using the QIAamp Viral RNA Mini Kit (Qiagen). Genomic regions, encoding the proteolytic cleavage site within the NDV F gene, as well as the region, encoding the inserted RABV G, were transcribed into cDNA and amplified, using the Transcriptor One-Step RT PCR Kit (Roche Applied Science, Mannheim, Germany). Sanger-sequencing (Sequencer 3130 Genetic Analyzer, Applied Biosystems, Foster City, CA, USA) was used to confirm virus identity.
8
Genotyping APE1 T1349G Polymorphism
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Genotyping assays to evaluate the APE1 T1349G polymorphism were performed by sequencing cDNA samples. The PCR amplification was carried out in the Veriti® Thermal Cycler (Applied Biosystems) by using the primers 5′-GCTTCGAGCCTGGATTAAGAA-3′ (forward) and 5′-GGCCTGCATTAGGTACATATGCT-3′ (reverse) to amplify the target fragment of APE1. PCR conditions were 94°C (10 min), followed by 40 cycles of 94°C (45 sec), 50°C (30 sec), and 72°C (30 sec), and subsequent final extension at 72°C (5 min). PCR products were purified by using GFX PCR DNA and Gel Band Purification kit (GE Healthcare Life Sciences, Chalfont, UK), according to the manufacturer's instructions. The cDNA sequencing was carried out on the Sequencer 3130 Genetic Analyzer (Applied Biosystems) and the results were analyzed with the Sequencher Program (Gene Codes Corporation, Ann Arbor, MI, USA) using the reference sequence from NCBI (NM_001244249.1).
9
Viral RNA Isolation and Identification
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Viral RNA was isolated from allantoic fluid using Trizol reagent (Invitrogen) according to the manufacturer's instructions. Virus identity was verified by RT-PCR using OneStep RT-PCR Kit (Qiagen) to amplify selected regions. Virus identity was approved by sequencing (Sequencer 3130Genetic Analyzer, Applied Biosystems).
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