Ecori

The duck LEAP-2/pCR2.1-TOPO vector was digested with the restriction enzymes EcoRI and BamHI (Promega, Madison, WI, USA). The protein expression vector pET32a (Novagen, Madison, WI, USA) was also digested with the same restriction enzymes. The digested fragments were purified from the agarose gel using the PureLink Quick Gel Extraction Kit (Invitrogen, USA) and were ligated using T4 DNA Ligase (Invitrogen, USA). The ligated vector and insert were transformed into One Shot BL21 (DE3) Chemically Competent E. coli (Invitrogen, USA) and sequenced. Positive clones were incubated at 37°C overnight on a shaking incubator at 225 rpm in LB broth with ampicillin (50 μg/mL). The bacteria culture was then induced for recombinant protein expression with 1 mM isopropyl-β-D-thiogalctopyranoside (USB Corporation, Cleveland, OH, USA) for 4 h at 28°C, and the bacteria were centrifuged at 5,000×g for 15 min. The duck LEAP-2 recombinant protein was extracted with B-PER Bacterial Protein Extraction Reagent (Thermo Scientific, USA) and purified using HisPur Cobalt Resin (Thermo Scientific, USA). Recombinant duck LEAP-2 was eluted using 250 mM imidazole and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting using 6× His-tag antibody (Thermo Scientific, USA).

Seven micrograms of oil palm genomic DNA extracts were digested in parallel by either EcoRI, HindIII or BamHI restriction enzymes (Promega) according to the manufacturer's instructions. The eletrophoretic separation of digestion products was performed as described previously [3] (link) before transfer on a GeneScreenPlus membrane (Perkin Elmer). DNA templates for probe synthesis were obtained through the PCR amplification of regions corresponding to a sequence located immediately upstream of the EgDEF1 gene (P1 probe), or to an internal section of each retroelement under study (P2 probe for the gypsy element, P3 probe for the copia element). The respective locations of the probes are illustrated in Figure 1A, probe sizes and primer sequences are given in Table S3. After purification using the Qiaquick kit (Qiagen), 25 ng of PCR product were radiolabeled with [α-³²P]dCTP using the Random Primer DNA Labeling System Kit (Invitrogen). Hybridizations were performed at 65°C overnight in a mix of 100 μl PerfectHybrid Plus buffer (Sigma) plus 0.4 μl herring sperm DNA (Promega) per cm² of membrane surface. Filters were then briefly washed at room temperature in 2× SSC, 0.1% (w/v) SDS, then at 65°C for 20 min in 2× SSC, 0.1% (w/v) SDS then at 65°C for 20 min in 1× SSC, 0.1% (w/v) SDS, before scanning the blots on a Typhoon 8600 Imager System (Amersham).

Genomic DNA was extracted directly from approximately 250 mg of the fresh faecal sample using the Microbiome DNA Purification Kit, Purelink (Fisher, California, USA) according to the manufacturer’s instructions. DNA was eluted in 100 μL elution buffer and the working stock stored at −20 °C. Genomic DNA was used for polymerase chain reaction (PCR) with primers targeting gene regions of Blastocystis, Cryptosporidium and Giardia according to previously described protocols [23 (link)] (Appendix A Table A1). The purified gel extracts were eluted, of which 1.5 μL was used for cloning with the pGEM-T easy vector system I (Promega, Madison, WI, USA). Between five and ten colonies per transformation were inoculated and grown overnight in 5 mL LB media. The plasmid DNA was extracted using the GeneJet Plasmid Miniprep Kit and clones were confirmed as positive using EcoRI (Promega) restriction digestion. Positive clones were sent for sequencing using the T7 and/or SP6 universal primers (Eurofins, Ebersberg Germany).

Transfection was performed using the Lipofectamine 2000 Transfection reagent (Invitrogen) according to the manufacturer's protocol. The eukaryotic expression vector pcDNA3.1(+) (Invitrogen) was used to generate the IHH expression plasmid. The IHH genomic sequence was amplified with PCR, digested with EcoRI and BamHI (Promega, Madison, WI, USA), and subcloned into the pcDNA3.1(+) vector.