The CRISPR/Cas9 system has been harnessed to create a simple, RNA-programmable method to mediate genome editing in mammalian cells. CRISPR/Cas9 editing generates insertions or deletions (indels) that can result in gene knockout. In most cases, cells have two copies of any given gene and indel mutations can be generated in either one or both alleles. The Guide-it Genotype Confirmation Kit provides a simple protocol to determine whether gene editing resulted in indels on one allele (monoallelic) or both alleles (biallelic) in singly isolated cells (clones), allowing for the identification of clones with desired mutations for further analysis. The kit is designed to be used in conjunction with the Guide-it sgRNA In Vitro Transcription Kit (Cat. No. 632635), which generates a target-specific guide RNA used in the genotype confirmation reaction. The sequence of the guide RNA used for the genotype confirmation reaction is identical to the guide RNA used for the initial genome editing experiment.

The Guide-it Mutation Detection Kit contains all the reagents needed for PCR-based identification of insertions or deletions generated during cellular non-homologous end joining (NHEJ) repair. The first step is the amplification of the putative target sequence directly from cells. This kit uses Terra PCR Direct Polymerase Mix and Buffer, so there is no need to extract genomic DNA from your cell population prior to amplification of your target sequence. The amplicon is then melted and hybridized to form the mismatched targets for cleavage by the Guide-it Resolvase. Sufficient material is provided for 100 amplification and cleavage reactions.