PCR-based analyses of S. cerevisiae erg11
− strains carrying the YEpNP-gERG11 and YEpNP-cCYP51 vectors. PCR analyses to confirm the presence of the kanMX gene at the ERG11 locus (panel A: primers CYP51ScExt.F and KanMX4.R2 and panel B: primers KanMX4.F2 and CYP51ScExt.R), the presence of the X. dendrorhous CYP51 gene (panel C: primers RTCYP51.F and CYP51.Rb), the presence of the ERG11 gene (panel D: gERG11.F and gERG11.R) and the absence of the ERG11 gene in the S. cerevisiae genome (panel E: gERG11.F and CYP51ScExt.R). Template DNA in each lane: S. cerevisiae parental diploid strain erg11 +/− (Lane 1), S. cerevisiae S288c strain (Lane 2), S. cerevisiae Sc-hCYP51 strain (Lane 3), S. cerevisiae Sc-hERG11 strain (Lane 4), X. dendrorhous UCD 67–385 strain (Lane 5), negative control without DNA (Lane 6). Molecular size marker Lambda/Hind III (Lane M: 23.1, 9.4, 6.6, 4.4, 2.3, 2.0 and 0.6 kb). A schematic diagram is included to represent the primer sets (shown in arrows) that were used. The UP and DOWN regions correspond to regions located 300 bp upstream and downstream of the S. cerevisiae ERG11 gene, respectively. Region KanMX4 corresponds to the geneticin (G418) resistance module and the pACT4 and tTDH3 regions correspond to the S. cerevisiae promoter and terminator region in YEpNP.