Purelink plasmid miniprep kit

After inspection of the sequencing electropherograms, two samples were selected that contained every DAZ family member-specific SFV in the region covered by Fragment I. The fragment was freshly amplified and cloned with Invitrogen’s TOPO TA Cloning Kit as described in the manufacturers’ protocol. For either sample, overnight cultures were grown from 30 white colonies. Plasmids were cleaned up using Invitrogen’s PureLink Plasmid Miniprep Kit. The concentration of the plasmid preparations was measured and adjusted to 100.0 ng/μl. The inserts of the preparations were sequenced to find out which DAZ family member they must have been derived from. Four plasmid preps with insert of different origins were chosen to assemble control DNA mixtures in order to mimic Fragment I amplified in samples containing two, four or six DAZ copies in various combinations. The mixtures presented several known variant ratios at every SFV position. The composition of the DNA mixtures is shown in S4 Table.

Oligonucleotide primers for sequencing the full length of the oyster NR sequences CgRXR, CgRAR and CgPPAR (S1 Table) were designed with Primer-Blast at NCBI [46 (link)] based on C. gigas genomic DNA data [47 (link)] for each gene (GenBank: CGI_10004075 (CgRXR); CGI_10028545 (CgRAR); CGI_10011509 (CgPPAR)). Total RNA was extracted from frozen whole adults and mixed embryo oyster individuals (embryo toxicity test). Extraction, DNA digestion, reverse transcription, and amplicon visualization and purification were performed as described previously [24 (link)]. Amplicons were obtained by RT-PCR under the following conditions: 95°C for 2 min, thirty cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 2 min, and a final extension at 72°C for 5 min, and cloned into a pGEM-T Easy vector (Promega). Vectors were purified using the PureLink Plasmid miniprep kit (Invitrogen), and were subsequently sequenced by Eurofins MWG Operon (Cologne, Germany). Each identified receptor sequence was confirmed by three independent successful cloning attempts.
The obtained coding DNA sequences (CDS) for all receptors (GenBank accession numbers: CgRXR1: KX590999; CgRXR2: KX591000; CgRAR: KX591001; CgPPAR: KX591002) were aligned to their associated genomic DNA sequence to identify isoforms and their intron/exon structure.

E-gene 346-bp fragments from DENV2-positive samples were cloned into a pGEM-T Easy TA cloning vector (Promega, Madison, WI, USA) and chemically transformed into DH5α Escherichia coli. White transformed colonies were randomly selected from LB/Amp/IPTG/X-gal agar plates and subjected to colony PCR to check for the presence of an insert. Using a Purelink Plasmid miniprep kit (Invitrogen), plasmid DNA was then isolated from overnight cultures of single positive clones grown in Luria–Bertani broth containing 100 µg ml⁻¹ ampicillin. Six to 10 clones from each human sample and 14 clones from each mosquito sample were sequenced using M13 universal primers. The DENV2 E gene sequences generated from both humans and mosquitoes were submitted to the DNA Data Bank of Japan (DDBJ) under accession numbers LC030023–LC030105.