Noti and xbai restriction enzymes

The cDNA sequences for human wild type α4 (NCBI Reference Sequence: NM_000744.5), wild type β2 (NCBI Reference Sequence: NM_000748.2), α4(R336H) (Chen et al., 2009 (link)) and β2(V337G) (Liu et al., 2011 (link)) were used to synthesize full-length cDNA for each subunit (Life Technologies, Grand Island, NY). All constructs were fully sequenced and confirmed to be identical to the published sequences for each subunit. Each nAChR subunit cDNA was removed from the pMA shuttle vector using Not I and Xba I restriction enzymes (New England Biolabs, Ipswich, MA) and ligated into the pCI mammalian expression vector (Promega Madison, WI) using T4 DNA ligase (Promega, Madison, WI). The constructs were transformed into NEB 5-α competent E. coli cells (New England Biolabs, Ipswich, MA) for larger-scale production of cDNA. DNA was isolated using QIAprep Spin Miniprep kits (Qiagen, Valencia, CA). To prepare for cRNA synthesis, cDNA clones of the α4, α4(R336H), β2 and β2(V337G) subunits were linearized with the restriction enzyme Swa I and treated with proteinase K (30min at 50°C), then purified using Qiagen’s PCR clean-up kit. cRNAs were transcribed using the T7 mMESSAGE mMACHINE™ High Yield Capped RNA Transcription Kit (Ambion, Austin, TX). cRNA purity was confirmed on a 1% agarose gel and the final product was sub-aliquoted and stored at −80°C.