Pcr and gel purification kits

We utilized our previously described pCI construct for all transient transfections^{12 (link)}. A gene encoding MR1B_RFP or MR1C_RFP was ligated into this construct using EcoRI and KpnI, to create pCI_MR1B_RFP or pCI_MR1C_RFP. Confirmatory sequencing was performed. Restriction enzymes and ligation kit were obtained from New England Biolabs (Ipswich, USA). PCR and gel purification kits were obtained from Qiagen (Venlo, Netherlands).

A tetracycline inducible promoter was designed with a CMV minimal promoter, a human ubiquitin promoter and a reverse transactivator sequence (ThermoFisher Scientific). The sequence was flanked with AscI and NotI restriction sites and was cloned into the plasmid PCI ASCI, which is our previously described PCI plasmid^{15 (link)} with an AscI cut site upstream of the CMV promoter. The new construct is designated PCI ASCI TET. MR1GFP was ligated into this construct using EcoRI and KpnI to create TET-MR1GFP. Confirmatory sequencing was performed. Restriction enzymes and ligation kit were obtained from New England Biolabs. PCR and gel purification kits were obtained from Qiagen. Transfection of TET-MR1GFP was done using an Amaxa Nucleofector, Kit T solution (Lonza). Each transfection reaction was done with 1e6 cells and 6 µg of TET-MR1GFP plasmid.