Abi big dye terminator kit v 3

Total genomic DNA was extracted from single whole mosquitoes using a NucleoSpin^® Tissue kit (Macherey-Nagel, Duren, Germany) according to manufacturer’s instructions. The fragment of mtDNA cytochrome c oxidase subunit 1 (cox1) gene was amplified using two sets of primers, 1454F (5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) and 2160R (5′-TAA ACT TCT GGA TGA CCA AAA AAT CA-3′); and 2027F (5′-CCC GTA TTA GCC GGA GCT AT-3′) and 2886R (5′-ATG GGG AAA GAA GGA GTT CG-3′), following the polymerase chain reaction (PCR) protocol explicitly detailed in Zhong et al. [31 (link)]. Aliquots of the PCR products were visualized on 1.5% agarose gels and successful amplifications were purified using ExosapIT^® (USB Co, Cleveland, OH, USA). All sequencing reactions were carried out in both directions using an ABI Big Dye Terminator Kit v.3.1 (Applied Biosystems, Warrington, UK) and analyzed on an ABI Prism 3500xL—Avant Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) at the Institut Pasteur du Laos sequencing facilities in Vientiane.

Genomic DNA was isolated from individual mosquitoes using the QIAamp® DNA Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. Approximately 650 bp of the COII gene and a 550-bp PCR product of the ITS2 region was amplified using primer pairs LYS-R (5′-ACTTGCTTTCAGTCATCTAATG-3′) and LEU-F (5′-TCTAATATGGCAGATTAGTGCA-3′) and ITS2-R (5′-TATGCTTAAATTCAGGGGGT-3′) and ITS2-F (5′-TGTGAACTGCAGGACACAT-3′), respectively. ITS2 was amplified in a PCR reaction volume of 25 µl with the following cycling parameters: 94 ℃, 2 min; then 94 ℃/30 s, 50 ℃/30 s, 72 ℃/40 s for 40 cycles; and a final extension at 72 ℃ for 10 min. COII was amplified in a PCR reaction volume of 25 µl with the following cycling parameters: 95 ℃, 5 min; then 95 ℃/1 min, 51 ℃/1 min, 72 ℃/ 2 min for 35 cycles; wiath a final extension 72 ℃ for 10 min. The PCR products were then analyzed by 1.5% agarose gel electrophoresis stained with GoldView (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), under UV transillumination. The sequencing reaction proceeded in both directions with the assistance of an ABI Big Dye Terminator Kit v.3.1 (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA); analysis was conducted using ABI Prism 3500xL-Avant Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific) in Shanghai (Sangon Biotech).

For some specimens, it was problematic to obtain the entire mitochondrial DNA using Illumina sequencing technology only. Consequently, we obtained small fragments of certain portions of the mitochondrial genome to complete the circular DNA molecule. In these situations, we amplified the target DNA using primers that were developed for specific regions (electronic supplementary material, table S5). PCR products were electrophoresed in 1.0% TBE agarose gels stained with GelRed Nucleic Acid Gel Stain (Biotium Inc., Hayward, USA). Sanger sequencing reactions [38 (link)] were carried out in one direction using ABI Big Dye Terminator Kit v.3.1 (PE Applied Biosystems, Warrington, England). Sequencing reactions were purified in Sephadex G50^® columns (GE Healthcare), analysed on an ABI Prism 3130—Avant Genetic Analyser (Applied Biosystems, Foster City, CA, USA), and edited using Sequencher^® for Windows v. 5.1. Sanger DNA fragments were assembled to the mitochondrial genome obtained using Illumina sequencing technology to complete the circular molecule.