Genomic DNA was extracted from entire specimens of B. solaris and P. paranygulgus using the DNeasy Blood & Tissue Kit (QIAGEN), following the manufacturer’s instructions. An 862–base pair (bp) region of the plastid rbcL gene, which encodes for the large subunit of the ribulose-1,5-biphosphate carboxylase/oxygenase, was PCR-amplified using Illustra PuReTaq Ready-To-Go PCR beads (GE Healthcare), and the primers and thermocycling conditions listed in table S1. Amplicons were visualized on 1.5% agarose gels stained with GelRed (Biotium) and enzymatically cleaned before sequencing with Illustra ExoProStar S (GE Healthcare). Clean amplicons were sequenced in 10 μl containing 1 μl of BigDye Terminator (BDT) v3.1 (Applied Biosystems), 2 μl of BDT buffer, 0.5 μM amplification primer, and 1 to 2 μl of PCR product. Cleaned sequencing products were run on an Applied Biosystems 3730S 48-capillary DNA analyzer by the Nucleic Acid Protein Service Unit at UBC. Resulting clean trace files were assembled into full sequences in Geneious v9.1.5 (32 (link)) and subjected to a BLAST (Basic Local Alignment Search Tool) search on the National Center for Biotechnology Information (NCBI) website (www.ncbi.nlm.nih.gov ) and an identification request in the Public Record Barcode Database on the BOLD (Barcode Of Life Data) website (www.boldsystems.org/index.php/ ) to verify the plastid’s taxonomic origin.
3730s 48 capillary dna analyzer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 3730S 48-capillary DNA analyzer is a genetic analysis instrument designed for high-throughput DNA sequencing. It utilizes capillary electrophoresis technology to separate and detect fluorescently labeled DNA fragments, enabling efficient and accurate genetic analysis.
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Lab products found in correlation
2 protocols using 3730s 48 capillary dna analyzer
1
PCR-based Barcoding of Organisms
2
Microsatellite Profiling of Salmonid Species
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Tissue samples were digested, and total genomic DNA was extracted using the DNeasy DNA blood and tissue extraction kit (Qiagen Inc., Valencia, CA, USA). Extracted DNA samples were stored at −20°C for use in multiplex polymerase chain reactions (PCR) using the Qiagen multiplex kit (Cat. No. 206145). DNA was amplified in 10 μL PCRs at 95°C for 15 min, 94°C for 30 sec, 35 cycles of 1.5 min at an annealing temperature of 55°C followed by 72°C for 1 min, and 60°C for 30 min. Microsatellite variation was assayed using primers labeled with fluorophores and a 3730S 48-capillary DNA Analyzer with GS 500 LIZ or 600 LIZ internal size standards (Applied Biosystems, Carlsbad, California, USA). Alleles were manually scored using the program GeneMapper (GeneMapper v.3.7, Applied BioSystems). We amplified 13 microsatellite loci isolated from other salmonid species: Atlantic salmon, Salmo salar (SSOSL456; Slettan et al. 1997 (link)); bull trout, Salvelinus confluentus (Sco200, Sco202, Sco215, Sco216, Sco220; DeHaan and Ardren 2005 ); chinook salmon, Oncorhynchus tshawytscha (OtsG83b, OstG253b; Williamson et al. 2002 ); Dolly Varden, Salvelinus malma (Smm-17, Smm-21, Smm-22, Smm-24; Crane et al. 2004 ); and rainbow trout, Oncorhynchus mykiss (OMM1105; Rexroad et al. 2002 (link)) (see May-McNally 2014 for full details).
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