Pcr 4 topo vector system

Degenerate primers were designed using MacVector v.11.1, NCBI Blast and known sbp2 truncated copy 7, 9 and 11 gene sequences from the Tx strains, to amplify and clone the three orthologous genes from the Australia T and D strain pairs (see Additional file 1: Table S1). Amplifications were carried out using the following PCR conditions: 1 cycle at 95 °C for 180 s; 39 cycles of 95 °C for 30 s, 54 °C for 30 s, 72 °C for 120 s and finally 1 cycle at 72 °C for 300 s (Sigma-Aldrich RedTaq reaction mix, MO, USA). The amplicons were gel purified, cloned using pCR®4-TOPO® vector system (Invitrogen, MA, USA) and sequenced (Qiagen Miniprep and Eurofins SimpleSeq DNA sequencing kit, Thermo Fisher Scientific, NV, USA). Subsequent strain-specific primers were designed to amplify the full-length of sbp2t7, sbp2t9 and sbp2t11 from cDNA of the Australian B. bovis strains. Validation of correct amplification was conducted by cloning and sequencing. Full-length of sbp2t7, sbp2t9 and sbp2t11 from the cDNA of Australian B. bovis strain pairs (T and D) were reported under the GenBank accession numbers MG430176, MG430177 and MG430178, respectively.

Candidate genes from the cytochrome P450 enzymes CYP314a1/shade (shd) and CYP302a1/disembodied (dib) and the 7,8 dehydrogenase neverland (nvd) known to be involved in the synthesis of ecdysteroids were identified in the salmon louse genome (www.Licebase.org) by homology to known sequences from insects and crustaceans. Sequences with the lowest e-value were chosen. Gene specific 5`and 3`RACE primers were designed (Table 1) and RACE was performed using the SMARTer^™RACE cDNA Amplification kit (Clontech, Mountain view, CA, USA) according to manufacturer’s recommendations (Sigma-Aldrich, St. Louis, MO, USA). Sub-cloning was performed using a pCR^®4-TOPO^® vector system (Invitrogen, Carlsbad, CA, USA) that were transformed into Escherichia coli TOP10 cells. Clones were verified by PCR with M13_f and M13_r primers (Table 1), grown overnight and purified using a Miniprep Nucleospin^® Plasmid Purification Kit (Macherey-Nagel, Duren, Germany). Plasmids were sequenced using a BigDye^® Terminator v3.1 Cycle sequencing kit (Applied Biosystems^®, Foster City, CA, USA) and analyzed in MacVector (MacVector Inc., NC, USA). Sequencing was performed by the Sequencing Facility at the Molecular Biological Institute, University of Bergen.