The mirKO production laboratory performs long range PCR (LR-PCR) across the shorter of the two homology arms with one generic primer within the targeting cassette and a locus specific primer outside the targeting vector.
Following expansion of the candidate targeted clones to archive plates; the LR-PCR for the shorter arm is repeated to confirm correct clone selection. LR-PCR across the longer homology arm is then performed. Amplification of appropriately sized PCR products at both ends indicates with high confidence that a replacement event has occurred.
However, we have found that even when the long arm PCR fails (especially if in all clones for a particular miRNA) the targeting event can still be correct.