PCR products were purified with ExoSAP-IT (USB Corporation, Santa Clara, CA, USA) by incubation at 37°C for 15 min, followed by enzyme inactivation for 15 min at 85°C, according to the manufacturer's instructions. The resulting purified fragments were sequenced using the ABI Big Dye Terminator Cycle Sequencing Ready Reaction kit v3.1 (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA, USA) and analyzed in an ABI PRISM 3130xl (Applied Biosystems).
Abi big dye terminator cycle sequencing ready reaction kit v3
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI Big Dye Terminator Cycle Sequencing Ready Reaction kit v3.1 is a DNA sequencing reagent kit. It is used for performing automated DNA sequencing analysis.
Automatically generated - may contain errors
Lab products found in correlation
Aligner, by CodonCode (3 mentions)
Abi prism 3130xl, by Thermo Fisher Scientific (2 mentions)
Exosap it, by Thermo Fisher Scientific (2 mentions)
Accuprep genomic dna extraction kit, by Bioneer (1 mentions)
Accuprep pcr purification kit, by Bioneer (1 mentions)
3730xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
5 protocols using abi big dye terminator cycle sequencing ready reaction kit v3
1
DNA Sequencing Using ABI Terminator Kit
2
Mitochondrial DNA Barcoding of Fish
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Genomic DNA was extracted from muscle tissue or pectoral fin clips using the AccuPrep Genomic DNA extraction Kit (Bioneer, Daejeon, Korea). Partial fragments of mitochondrial DNA cytochrome c oxidase subunit I (mtDNA COI) were amplified using VF2 and FishR2 (5′-TCW ACC AAC CAC AAA GAY ATY GGG AC-3′ ) primers [28 (link)]. We carried out the PCR under the following conditions: initial denaturation for 5 min at 95°C, 35 cycles (denaturation for 1 min at 95°C, annealing for 1 min at 52°C, extension for 1 min at 72°C) and final extension for 5 min at 72°C. The amplified PCR product was electrophoresed in 1% agarose gel and visualized by an UV illuminator. PCR products were purified the using AccuPrep® PCR purification kit (Bioneer, Korea) and directly sequenced using the ABI BigDye terminator cycle Sequencing Ready Reaction Kit v.3.1 (Applied Biosystems Inc., USA). The mtDNA COI sequences were aligned using ClustalW [29 (link)] in BioEdit v.7 [30 ]. The mtDNA COI Sequences were registered to the DDBJ / EMBL / GenBank nucleotide sequence databases (accession numbers KY773707–KY773908).
3
Sanger Sequencing of PCR Products
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Purified PCR products were subjected to bidirectional Sanger sequencing using a dideoxy chain termination protocol with ABI BigDye Terminator Cycle Sequencing Ready Reaction Kit v3.1 (Applied Biosystems, Foster City, CA, USA) and a programmable thermal cycler (Veriti, ABI, USA), as per [20] .DNA sequences were assembled using the computer program CodonCodeAligner (CodonCode Corporation, USA). Sequences were deposited in Genbank (Table 1 ).
4
PCR Product Purification and Sequencing
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PCR products were purified with ExoSAP-IT (USB Corporation, Santa Clara, CA, USA) according to the manufacturer's instructions and sequenced with the ABI Big Dye Terminator Cycle Sequencing Ready Reaction kit v3.1 (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA, USA). Fragments were analyzed in an ABI PRISM 3130xl (Applied Biosystems). Sequences were aligned using Geneious v.5.5 (created by Biomatters, available from http://www.geneious.com/ ).
5
Automated Sanger Sequencing of PCR Products
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Purified PCR products were sequenced using a dideoxy chain termination protocol with ABI BigDye Terminator Cycle Sequencing Ready Reaction Kit v 3.1 (Applied Biosystems, Foster City, CA, USA) and a programmable thermal cycler (Veriti, ABI, USA). Two reactions were used to amplify both strands (i.e., one with forward primer and the other with reverse primer). In order to eliminate unincorporated dye terminators, SDS (0.2% final concentration) was added to the cycle sequencing reaction products and heat treated at 98°C for 5 minutes, followed by 25°C for 10 minutes. Reactions were then purified by Centri-Sep spin column (Applied Biosystems, Foster City, CA, USA). Purified extension products were vacuum dried and DNA sequencing was performed (Applied Biosystems 3730xl Genetic Analyzer, Foster City, CA, USA). DNA sequences were captured as color-coded electropherograms and were assembled using computer program CodonCodeAligner (CodonCode Corporation, USA). Original sequences are available from the first author upon request and had been submitted to Genbank, with accession numbers as provided in Table 1 .
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