Hey Everyone,

I have a CRISPR-Cas9 rat model that we genotype to verify heterozygotes. The company that designed the rats gave us the primer sequences (2 for genomic and 2 for knockout) and we recently discovered that it will only give us bands for "Wild type" or genomic plus the deleted sequence when all four primers are combined into one mixture. The primer sequences for the genomic allele match up to the proper allele when I run it through BLAST. If we run two PCR cycles; one for knockout screening and the other for genomic screening; we see the correct band on the knockout screen but NOTHING on the wild type screen. I did this because we used a new thermocycler and thought something was wrong with our recipe (turned out I needed to order new primers). But now even with new primers, it only looks successful when all primer are together.