The COI gene and D-loop region were amplified by PCR using the specific primers (Table 1 ). All PCR reactions were carried out in an ABI2720 Thermal Cycler (Applied Biosystems, USA) with a 20 µL reaction including 10 µL of 2 × Taq Master Mix (Taraka, Dalian, China), 0.5 µL of each primer, and about 50 ng template DNA. The thermal cycling for PCR amplifications is also listed in Table 2 . There was a negative control in each round of PCR to check the contamination, and all negative controls had no products. The PCR products of each sample were detected by electrophoresis on a 1.5% agarose gel. The bright main strip was purified and recovered using the QIAquick Gel Extraction Kit (Qiagen, GER). The purified PCR products were sequenced with an ABI 3730 XL automatic sequencer (Perkin-Elmer, Waltham, MA, USA).
Abi 3730xl automatic sequencer
Manufactured by PerkinElmer
Sourced in United States, China
The ABI 3730XL automatic sequencer is a high-throughput DNA sequencing instrument designed for genomic research and analysis. It features a 96-capillary array and utilizes the Sanger sequencing method to efficiently generate DNA sequence data.
Automatically generated - may contain errors
Lab products found in correlation
Amplitaq fs dna polymerase, by Thermo Fisher Scientific (2 mentions)
Extaq buffer mg2 free, by Takara Bio (2 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Abi 2720 thermal cycler, by Thermo Fisher Scientific (1 mentions)
Dneasy tissue kit, by Qiagen (1 mentions)
Ex taq polymerase, by Takara Bio (1 mentions)
Abi prism bigdye terminator cycle sequencing ready reaction kit, by Thermo Fisher Scientific (1 mentions)
Mycycler gradient thermocycler, by Bio-Rad (1 mentions)
3 protocols using abi 3730xl automatic sequencer
1
COI and D-loop PCR Amplification and Sequencing
2
Phylogenetic Analysis of Rattus using mtDNA
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Genomic DNA was extracted from muscle or tail tips using a DNeasy Tissue Kit (QIAGEN). Three mtDNA fragments (COI: 681 bp, D‐loop: 1048 bp, and cyt b: 1143 bp), extensively used in phylogenetic analyses of Rattus, were amplified separately with primers listed in Table S2 .
The PCR was done in a 50 μl reaction volume, including 5 μl of 10× EXTaq buffer (Mg2+ Free; Takara Biotech), 10 mM of each dNTP, 75 mM MgCl2, 10 μM of each primer, 1.5 U EXTaq polymerase (Takara Biotech) and ~20–50 ng total genomic DNA. Amplifications were done as described (Pagès et al., 2010 (link)) with some modifications of annealing temperatures. Each round of PCR included one negative control to avoid contamination, and the negative control yielded no product. The PCR products were purified with a gel extraction kit (Sangon BioMedical), and directly sequenced (both directions) with an ABI 3730XL automatic sequencer (Perkin‐Elmer) using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (with AmpliTaq DNA polymerase FS, Applied Biosystems).
The PCR was done in a 50 μl reaction volume, including 5 μl of 10× EXTaq buffer (Mg2+ Free; Takara Biotech), 10 mM of each dNTP, 75 mM MgCl2, 10 μM of each primer, 1.5 U EXTaq polymerase (Takara Biotech) and ~20–50 ng total genomic DNA. Amplifications were done as described (Pagès et al., 2010 (link)) with some modifications of annealing temperatures. Each round of PCR included one negative control to avoid contamination, and the negative control yielded no product. The PCR products were purified with a gel extraction kit (Sangon BioMedical), and directly sequenced (both directions) with an ABI 3730XL automatic sequencer (Perkin‐Elmer) using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (with AmpliTaq DNA polymerase FS, Applied Biosystems).
3
mtDNA Sequencing of Egretta garzetta
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The E. garzetta mtDNA was obtained by polymerase chain reactions (PCR) using 28 primer sets reported by Sorenson et al. (1999) (link). The PCR products for each set of primers were < 1,500 bp in size and all fragment sequences overlapped each other by at least 200 bp. PCR amplifications were done with a Mycycler Gradient thermocycler (Bio-Rad) in a final volume of 50 μL, including 5 μL of 10x EXTaq buffer (Mg2+-free; Takara Biotech, Dalian, China), 2.5 mM of each dNTP, 75 mM MgCl2, 10 μM of each primer, 1.5 U of EXTaq polymerase (Takara of Biotech, Dalian, China) and approximately 20–50 ng of total genomic DNA. The reaction included an initial denaturation at 94 °C for 3 min, followed by 35 cycles consisting of denaturation at 94 °C for 10 s, annealing at 50–56 °C for 30 s and extension at 72 °C for 2 min, with a final extension at 72 °C for 10 min. There was a negative control in each round of PCR to check for contamination. The products were electrophoresed on 1.5% agarose gels staining with ethidium bromide and visualized by ultraviolet transillumination. The PCR products were purified with a gel extraction kit (Sangon BioMedical, Shanghai, China) and directly sequenced (both directions) with an ABI 3730XL automatic sequencer (Perkin-Elmer) using an ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit (with AmpliTaq DNA polymerase FS, Applied Biosystems).
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