This assay does not work well without the use of a Hotstart Taq polymerase.
When using the two primers flanking the loxp sites (10858/10859), the assay may not reliably amplify and separate both fragments from heterozygous mutant mice. Therefore we have included a mutant specific (loxp) primer that can be used to amplify from the mutant allele (oIMR9042).
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.