Be aware: In multiplex reaction the targeted mutant band may be faint in heterozygotes and mistyped as homozygous wild type.
This assay does not work well without the use of a Hotstart (We are using Taq Start Antibody mixed 1:1 with Taq polymerase).
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 = 753 bp
Heterozygote = 572 bp and 753 bp
Wild type = 572 bp
Separated by gel electrophoresis on a 1.5% agarose gel.