Embryonic PCR Protocol
Embryonic PCRs are very valuable in determining the cleavage efficiency of a pair of gRNAs as well as HDR efficiency. These reactions can be done on embryos 24 hours after injections for rapid feedback on the likelihood a particular strategy will work.
Testing a pair of gRNAs: Design oligos flanking the two cut sites (i.e. spanning a deletion created by cleavage at both sites). We generally pick priming sites ~200-300 bp outside of the target sites such that 400-600 bp band will indicated cleavage at both sites.
Testing HDR efficiency: Design one oligo specific to the marker, insertion, or allele used in your HDR strategy. For example, when using pHD-DsRed-attP, we use primers specific to the 3xP3-DsRed-SV40 portion of the vector. Design a second oligo specific to the target locus but outside of the homology arms such that the PCR is specific for integration at the target locus. For strategies involving two distinct homology arms, as with pHD-DsRed-attP, we use two separate PCR reactions to test each side of the intended HDR event.
Preparing embryonic genomic DNA