Every cell has two alleles of most genes. CRISPR/Cas9 gene editing can introduce insertion or deletion mutations (indels) on either one or both alleles of a given gene. The Guide-it Genotype Confirmation Kit provides a simple method to determine if a given clone has mutations in one allele (monoallelic), both alleles (biallelic), or is unchanged (wild type). The protocol involves amplification of the target site from cells treated with Cas9 and a single guide RNA (sgRNA). The resulting amplicon is used in an in vitro cleavage reaction with recombinant Cas9 and the same sgRNA. The genotype can be determined by resolving the cleavage products on an agarose gel.
Using this kit after gene editing expedites the process of screening a large number of clones for those with the desired genotype. Each kit contains sufficient reagents for 100 extraction, amplification, and cleavage reactions.