on a 1.5% agarose gel, by comparing bands of the expected ligation product size against a standard curve of known DNA concentration (ten fivefold serial dilutions of 2 µg/µl−1 micrococal nuclease DNA) to control for possible unequal efficiency of ligation and to ensure that equal amounts of correctly ligated plasmids were used in the transfections. The ligated plasmid was directly transfected into HEK293 cells and was not subcloned to avoid loss of methyl groups from CG dinucleotides during growth in E. coli, which do not express CG DNA methyltransferases. For deletion constructs of the exon 1F NR3C1 promoter plasmids were prepared and ligations verified in the same manner as described above, except that oligonucleotides for NR3C1 promoter amplification were designed that incorporated HindIII and EcoRV restriction sites, obviating the need for ligation into PCR2.1 vector before ligation into the pGEM-luc vector (also see Supplementary Methods).
