Abi 3730 automated dna sequencer

We extracted genomic DNA from dried leaf tissue using the CTAB method (Doyle and Doyle, 1987 ). We amplified the chloroplast trnL-trnF intergenic spacer and nrITS regions using the universal primers trn-c and trn-f (Taberlet et al., 1991 (link)), and ITS4 and ITS5 (White et al., 1990 ), respectively. Two low-copy nuclear genes (tpi and cam), which encode triose phosphate isomerase and calmodulin, respectively, were PCR-amplified and sequenced using the primer sequences from Dai et al. (2012) (link) and Chao et al. (2014) (link), respectively. We purified the PCR products using the Pearl Gel Extraction Kit (Pearl Bio-tech, Guangzhou, China) and then directly sequenced them on an ABI 3730 DNA automated sequencer with the BigDye chemistry (Applied Biosystems, Foster City, CA, USA). For sequences with more than one polymorphic site and insertion/deletion polymorphisms, cloning-sequencing was conducted to phase the haplotypes. We conducted ligation reactions with a pMD18-T&A cloning kit (Takara, Dalian, China) and selected eight positive colonies for each individual for sequencing. We deposited all the sequences in GenBank with accession numbers KY798014–KY798110.

In this study, PCR amplification and Sanger sequencing were conducted for multiple purposes: (1) to verify the highly differentiated region in M. normale identified by Pool-seq, (2) to determine the copy number of the myb114 gene in M. normale and other Melastoma species, (3) to carry out population genetics analyses of myb114 and six surrounding segments in M. normale, and (4) to reconstruct the phylogenetic tree of Melastoma species in China based on sequences of the myb114 gene. All the primers were designed based on the reference genome of M. candidum, and primer sequences are listed in Table S12. PCR was conducted in a total volume of 25 µL with KOD FX DNA polymerase (TOYOBO, Osaka, Japan). The purified PCR amplification products were then directly sequenced on an ABI 3730 DNA automated sequencer with the BigDye chemistry (Applied Biosystems, Foster City, CA, USA). For sequences that contained multiple polymorphic sites, clonal sequencing was performed using the pMD-18 T Vector Kit (Takara, Dalian, China). Eight positive clones were sequenced to phase the haplotypes of each sample in DNASP. These sequences have been deposited in GenBank with the accession numbers MK618466-MK618508, MT010136-MT010215, and ON565433.

Total RNA was isolated from patient whole blood using the ipureTRizol blood RNA kit (IGE, Guangzhou, China). First-strand cDNA was synthesized from total RNA with an oligodT primer (First strand cDNA synthesis Kit,IGE, Guangzhou, China). Primers were designed and PCR was carried out to amplify the whole exon 8 (F 5′-CTTCTCTGTGGTGGGGATGG-3’and R 5′-TGCCCACGGTAATCTCAAAC-3′). The PCR products were gel purified (QIAquick gel extraction kit; QIAGEN) and directly sequenced on an ABI 3730 Automated DNA Sequencer (PE Applied Biosystems).

Fossorial tadpoles (N = 26) believed to be Micrixalus herrei, were euthanized in tricaine methanesulfonate (MS-222). A specimen was preserved in absolute ethanol, and stored at –20°C in the Systematics Lab, University of Delhi (SDBDU) for species verification. DNA was extracted from ethanol-preserved tail muscle tissue using a standard protocol [27 ]. A 540 bp fragment of the mitochondrial 16S ribosomal RNA was PCR-amplified following primers and protocol of Simon et al. [28 ]. Sanger sequencing was performed on both strands using an ABI 3730 automated DNA sequencer (Applied Biosystems). Sequences were assembled and edited in ChromasPro v1.34 (Technelysium Pty Ltd.). Preliminary identification of the taxon was done by conducting BLAST searches against a near-complete database of sequences of Micrixalus species. Subsequently, the generated sequence was compared against a dataset consisting of Micrixalus herrei (KJ711301, closest hit of the BLAST search) and other micrixalid species that were reported to occur in the same area [8 ], M. fuscus (KJ711283), M. mallani (KJ711324) and M. sali (KJ711354). ClustalW, as implemented in MEGA 5.0 [29 (link)], was used to align the sequences. MEGA was also used to calculate uncorrected pairwise divergences. Newly generated sequence was deposited in GenBank under the accession number KU833220.

Bacterial genomic DNA was extracted from pure culture with the fast spin kit
(Invitrogen) following the manufacturer's instructions. Amplification of 16S
rRNA gene was performed in 50 μL of reaction mixture containing 0.25 mM each
primer of 27f (5′-GTTTGATCCTG GCTCAG-3′) and 1492r (5′-TACCTTGTTACGACTT-3′), 0.2
mM dNTP, 1.5 mM MgCl₂, 5 μL of Taq buffer, and 5 U Taq DNA polymerase
(Invitrogen, USA), 10–20 ng template DNA. PCR was then performed on a
thermalcycler under the following conditions: 95 °C for 5 min, 35 cycles of 50 s
at 95 °C, 50 s at 45 °C and 1.5 min at 72 °C, followed by a final extension for
10 min at 72 °C. The PCR products were visualized on an agarose gel, and the
bands with the corrected size were excised and purified using the Wizard SV gel
purification protocol (Promega, USA). The partial 16S rRNA fragment was
sequenced on an ABI 3730 automated DNA sequencer (Applied Biosystems).

Adult specimens of P. tibialis were collected from alfalfa in Yuzhong County, Lanzhou City, Gansu Province, China, on 5 June 2014. Samples and voucher specimens are deposited in the State Key Laboratory of Grassland Agro-Ecosystem, College of Pastoral Agricultural Science and Technology, Lanzhou University in Lanzhou, China. All specimens were initially preserved in 100% ethanol in the field, and transferred to −20 °C until used for DNA extraction. The total genomic DNA was extracted from thorax muscle of a single specimen using the insect genome DNA extraction kit (Omega, Norcross, GA, USA), according to the manufacturer’s protocols. Polymerase chain reactions (PCRs) were performed with the LA PCR Kit (TaKaRa, Shiga, Japan) following the manufacturer’s recommendations. The primers and their sources are presented in Table S1. PCR products were electrophoresed in 1.5% agarose gel, purified with an EasyPure PCR Purification Kit (TransGen Biotech, Beijing, China), and then both strands were sequenced with primer walking on an ABI 3730 automated DNA sequencer (Applied Biosystems, Carlsbad, CA, USA).
Failed and unsatisfactory sequencing fragments were cloned into the pEASY-T1 vector (TransGen Biotech, Beijing, China), and then conducted bidirectional sequencing.

Total genomic DNA was extracted from adult and tadpole tissue samples using Qiagen DNeasy tissue kit, following the manufacturer’s protocols. Based on availability of GenBank data, the following gene fragments were PCR-amplified using previously published primers: two mitochondrial (mt) genes—ribosomal subunit 16 S rRNA (16 S, ∼560 bp, primer set 16Sar and 16Sbr)⁵⁵ and cytochrome oxidase I (CO1, ∼650 bp, primer set Chmf4 and Chmr4)^{56 (link)}, and four nuclear (nu) genes—brain-derived neurotrophic factor (BDNF, ∼700 bp, primer set BDNF.Amp.F1 and BDNF.Amp.R1)^{36 (link)}, histone H3 (His3, ∼330 bp, primer set H3F and H3R)⁵⁷ , seven in absentia homolog 1 (SIA1, ∼399 bp, primer set SIA1 and SIA2)⁵⁸ and tyrosinase (Tyr, ∼530 bp, primer set TyrC and TyrG)^{15 (link)}. Sequencing was performed on both strands using the BigDye terminator cycle sequencing kit on ABI 3730 automated DNA sequencer (Applied Biosystems). Nucleotide sequences were checked and assembled in ChromasPro v1.34 (Technelysium Pty Ltd.), and deposited in the Genbank under accession numbers MK285340–MK285351.