Guide-itTM Indel Identification Kit

Description

The Guide-it Indel Identification Kit is used for characterization of insertions and deletions indels generated by gene editing tools, such as CRISPR/Cas9. This kit contains all of the components needed to amplify, clone, and prepare modified target sites for DNA sequence analysis. This kit uses TerraPCR Direct to amplify targets directly from crude genomic DNA extracts. The resulting pool of fragments, which may contain a variety of indels, are cloned into a prelinearized pUC19 vector using the In-Fusion cloning system. Colony PCR of individual clones using Terra PCR Direct followed by DNA sequencing allows indel characterization. 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 Back The Guide-it Indel Identification Kit provides a complete workflow for identifying the variety of insertions and deletions indels introduced by nuclease-based genome editing.The protocol uses direct PCR to amplify a genomic DNA fragment 500 to 700 bp containing the target site directly from crude cell lysates step 1 . The resulting amplified fragments contain a pool of edited target sites from individual cells. These PCR products are cloned directly into a pre-linearized pUC19 vector using the In-Fusion Cloning system step 2 . After transformation of an optimizedE. colistrain, colony PCR is used to amplify the target site from the plasmid step 3 . DNA sequencing is then used to identify the different indels generated at the targeted genomic site step 4 Back 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 .