Two different PCR strategies were used for genotyping the BptfΔexon2 line. The primers used in the first strategy are as follows, Primer A (JL648), Primer B (JL649), and Primer C (JL652) (see Table S3 for primer sequences). PCR reactions were performed in 25 µl volumes with 20 mM Tris-HCL (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM each dNTP, 0.2 µM each primer, 1 Unit Taq Polymerase (Invitrogen). Reactions were heated for 3 min at 94°C, then cycled for 35 cycles at 30 sec at 94°C, 30 sec at 60°C, and 45 sec at 72°C, followed by one cycle for 2 min at 72°C. PCR products were resolved on 1.5% agarose gels. The genotype of embryos was confirmed by the presence of a 250 bp Bptf, 325 bp BptfFloxed and BptfFloxedNeo or 550 bp BptfΔexon2 band.
