The primers used in the second method are as follows, Primer C (JL651), Primer E (JL655), and Primer F (JL662) (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 65°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 375 bp Bptf, 475 bp BptfFloxed or 650 bp BptfΔexon2 bands.
