Guide-itTM Mutation Detection Kit

Description

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 TerraPCR 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. Our products are to be used forResearch Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Back The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells.The CRISPR/Cas9 system relies on a single guide RNA sgRNA directing the Cas9 endonuclease to induce a double strand break at a specific target sequence three base-pairs upstream of a PAM sequence in genomic DNA. This DNA cleavage can be repaired in one of two ways: 1 nonhomologous end joining, NHEJ resulting in gene knockout due to error-prone repair orange , or 2 homology-directed repair HDR , resulting in gene knockin due to the presence of a homologous repair template purple . Back Back Comparison of the Guide-it and Surveyor assays for detecting mutations in mammalian cells.293T cells were transfected with plasmids encoding Cas9 and a sgRNA specific for theAAVS1locus. Transfected cells were harvested 48 hr post-transfection and mixed with untransfected cells at varying ratios. An amplicon containing the targetedAAVS1locus was generated using Terra Direct Polymerase Mix, and the PCR products were purified and cleaved using either Guide-it Resolvase or the Cel1 enzyme Surveyor assay . Mutations were easily discernable when using the Guide-it kit. In contrast, the Surveyor Assay showed considerable smearing, making it difficult to determine cleavage efficiency and reducing the ability to detect low levels of mutation. Back Successful knockout ofAcGFP1in HT1080 cells using the CRISPR/Cas9 system.Panel A. Schematic of theAcGFPDNA sequence and the location of sgRNAs tested and primer placement for the mutation detection assay. HT1080 cells containing a single copy ofAcGFP1were transfected with 1.5 mug of plasmid DNA for Cas9 expression and 1.5 mug of a plasmid harboring one of two sgRNAs T1 or T2 using Xfect Transfection Reagent. The cell population was assayed 6 days post-transfection for cleavage efficiency and loss of fluorescence. Panel B. Using the Guide-it Mutation Detection Kit, cleavage products were detected for both sgRNAs, indicating that both CRISPRs successfully disrupted theAcGFP1locus. Panel C. TheAcGFP1disruptions were functionally relevant, as a subpopulation of non-fluorescent cells could be detected by FACS. Back