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 = 2500 bp and 300 bp
Heterozygotes = 2800 bp and 2500 bp and 300 bp
Wild type = 2800 bp (uncut)
Separated by gel electrophoresis on a 1.5% agarose gel.**