Confirmation of the loxP site downstream of the critical exon can be achieved by several methods.

The TRPCR used by the EUCOMM screen uses  universal primers to amplify across the critical exon region. Presence of a band (when accompanied by the proper controls) can be used as confirmation of the loxP site.

In some cases the region to be amplified may be too large for the standard PCR reaction, in which case a gene-specific primer can be used in conjunction with one of the universal primers or two gene specific primers can be used. The latter method will produce two bands of different sizes in heterozygotes, although this may be difficult to resolve on some gel systems.