This assay does not work well without the use of a Hotstart (We are using Taq Start Antibody mixed 1:1 with Taq polymerase).
Use Perfect Match© (Stratagene) added before Taq to delete non-specific bands.
The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX).
To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility.
Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.
Mutant = 427 bp
Heterozygote = 366 bp and 427 bp
Wild type = 366 bp
Separated by gel electrophoresis on a 1.5% agarose gel.