Hifi dna assembly cloning system

The cDNAs used were Ca_v1.4 (GenBank: A930034B14), β_2a (GenBank: NM_053851), β_2X13 (GenBank: KJ789960), and α₂δ−4 (GenBank: NM_172364) in pcDNA3.1 (Lee et al., 2015 (link)). To create the Ca_v1.4 cDNA with the G369i mutation, a codon insertion corresponding to the glycine insertion at residue G369 was generated with the HiFi DNA Assembly Cloning System (New England Biolabs) using Ca_v1.4 cDNA as the PCR template and the primers a1FbegNotF: 5'-TATATGCGGCCGCCCACCATGGATTAC-3’, Fr1G369insR: 5'-CTTAGGACACCTCCAAGCACAAGGTTGAGG-3’, Fr2G369insF: 5'-TTGGAGGTGTCCTAAGCGGGGAGTTC-3’, a1FBSrGIr: 5'-TTTAGGCAGCGTGTACAGCTAGCCATGGTCC-3’. All constructs were verified by DNA sequencing before use. Human embryonic kidney 293 T (HEK293T) cells (American Type Culture Collection, Manassas, Virginia) were cultured in Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific, Waltham, MA) with 10% FBS (VWR) at 37°C in 5% CO₂. At 70–80% confluence, the cells were co-transfected with cDNAs encoding mouse Ca_v1.4 (1.8 μg) β_2X13 (0.6 μg), α₂δ−4 (0.6 μg), and enhanced GFP in pEGFP-C1 (0.1 μg) using FuGENE six transfection reagent (Promega, Madison, WI) according to the manufacturer’s protocol. Cells treated with the transfection mixture were incubated at 37°C for 24 hr. Cells were then incubated at 30°C for an additional 24 hr before beginning experiments.