Pcdna3.1 v5 his topo ta expression vector

The pcDNA™3.1/V5-His TOPO^® TA Expression vector (Invitrogen, Paisley, UK) was used to clone amplified RT-PCR products. The primers used for the FL Oct-2 cDNA are (5′–3′):
And for the anti-sense Oct-2 fragment (5′ – 3′):

Full length sequences for SchgrETHR and SchgrETHpre were found in the in-house S. gregaria transcriptome database. The complete ORF was PCR-amplified using fifth instar S. gregaria head cDNA and Q5^® High-Fidelity DNA Polymerase (New England Biolabs). A first PCR was performed with the primers listed in Table 1, after which the PCR product was purified using Sigma-Aldrich’s GenElute™ PCR Clean-Up kit, and subsequently used as a template in a nested reaction using the second primer set (Table 1). Amplicons were analysed on a 1.2% agarose gel, purified using the GenElute Gel Extraction Kit (Sigma-Aldrich), cloned into a pcDNA3.1/V5-His-TOPO TA expression vector (Invitrogen) and transformed into One Shot TOP10 chemically competent E. coli cells (Invitrogen). The sequence of the insert was confirmed by Sanger sequencing. Colonies harbouring the correct receptor insert were grown overnight in 100 ml Luria-Bertani medium and plasmid DNA was isolated using Qiagen’s EndoFree Plasmid Maxi Kit.