Standard end-point PCR genotyping cannot distinguish homozygous (Tg/Tg) from hemizygous/heterozygous (Tg/0) transgenic mice, and labs adapting a JAX protocol to different equipment see inconsistent results.

Resolve zygosity and cross-lab protocol issues: 1) To determine zygosity, use quantitative Southern blot, quantitative PCR (qPCR), or progeny testing: mate the Tg/? mouse to a wild-type mate and genotype the offspring — if any offspring lack the transgene, the parent is hemizygous (Tg/0). 2) When adapting a protocol to different Taq polymerase or equipment, adjust the elongation time to match the specific polymerase's processivity rather than reusing JAX's published timing as-is. 3) Run singleplex reactions instead of multiplex when troubleshooting inconsistent bands, and always include positive, negative, and no-template controls. 4) Use melting-curve analysis (Tm = 2[A+T] + 4[G+C]) to confirm PCR product identity/size without running a gel.