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Guide-it Mutation Detection Kit

A faster and simpler method than the surveyor assay for identifying mutations such as insertions or deletions in mammalian cells

The Guide-it Mutation Detection Kit contains all the reagents needed for PCR-based identification of insertions or deletions generated in the genome of cells treated with engineered nucleases such as zinc-finger nucleases (ZFNs), transcription-activator-like effector nucleases (TALENs), or CRISPR/Cas9. 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. Agarose gel electrophoresis can be used to visualize the cleavage products.

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Cat. #	Product	Size	Price
631448	Guide-it™ Mutation Detection Kit	25 Rxns	USD $248.00
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. Notice to purchaser Our products are to be used for Research 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. Documents Components You May Also Like Image Data Back The Guide-it Mutation Detection Kit is used to confirm the presence of mutations in genomic DNA The Guide-it Mutation Detection Kit is used to confirm the presence of mutations in genomic DNA. In the first step your target sequence is amplified directly from your target cells using the Terra PCR Direct Polymerase included in the kit, so there is no need to extract and purify 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 that can be cleaved by the Guide-it Resolvase. Back Comparison of the Guide-it and Surveyor assays for detecting mutations in mammalian cells 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 the AAVS1 locus. Transfected cells were harvested 48 hr post-transfection and mixed with untransfected cells at varying ratios. An amplicon containing the targeted AAVS1 locus 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 of AcGFP1 in HT1080 cells using the CRISPR/Cas9 system *Successful knockout of AcGFP1* in HT1080 cells using the CRISPR/Cas9 system.** Panel A. Schematic of the AcGFP DNA sequence and the location of sgRNAs tested and primer placement for the mutation detection assay. HT1080 cells containing a single copy of AcGFP1 were transfected with 1.5 μg of plasmid DNA for Cas9 expression and 1.5 μg 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 the AcGFP1 locus. Panel C. The AcGFP1 disruptions were functionally relevant, as a subpopulation of non-fluorescent cells could be detected by FACS. Back 631448: Guide-it Mutation Detection Kit
631443	Guide-it™ Mutation Detection Kit	100 Rxns	USD $531.00
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. Notice to purchaser Our products are to be used for Research 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. Documents Components You May Also Like Image Data Resources Back The Guide-it Mutation Detection Kit is used to confirm the presence of mutations in genomic DNA The Guide-it Mutation Detection Kit is used to confirm the presence of mutations in genomic DNA. In the first step your target sequence is amplified directly from your target cells using the Terra PCR Direct Polymerase included in the kit, so there is no need to extract and purify 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 that can be cleaved by the Guide-it Resolvase. Back Comparison of the Guide-it and Surveyor assays for detecting mutations in mammalian cells 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 the AAVS1 locus. Transfected cells were harvested 48 hr post-transfection and mixed with untransfected cells at varying ratios. An amplicon containing the targeted AAVS1 locus 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 of AcGFP1 in HT1080 cells using the CRISPR/Cas9 system *Successful knockout of AcGFP1* in HT1080 cells using the CRISPR/Cas9 system.** Panel A. Schematic of the AcGFP DNA sequence and the location of sgRNAs tested and primer placement for the mutation detection assay. HT1080 cells containing a single copy of AcGFP1 were transfected with 1.5 μg of plasmid DNA for Cas9 expression and 1.5 μg 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 the AcGFP1 locus. Panel C. The AcGFP1 disruptions were functionally relevant, as a subpopulation of non-fluorescent cells could be detected by FACS. Back The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells 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 631443: Guide-it Mutation Detection Kit

Overview

Simple method to identify insertions or deletions in mammalian cells
Faster and more efficient than the surveyor assay; amplify directly from your target cells
Complete kit: Guide-it Resolvase, Terra PCR Direct Polymerase, and all buffers included
Value: 100 reactions per kit; also available in a 25-reaction size for small-scale genome editing projects
Perfect for confirming sequence changes downstream from Cas9/CRISPR, TALENs, or zinc-finger nuclease studies

More Information

Applications

Confirmation of mutations caused by genome editing technologies, including CRISPR/Cas9, TALENs, or zinc-finger nucleases
Detecting insertions and deletions in mammalian genomic DNA

Components

Extraction Buffer 1
Extraction Buffer 2
Guide-it Resolvase
Terra PCR Direct Polymerase Mix (1.25 U/µl)
2X Terra PCR Direct Buffer (with Mg²⁺, dNTP)
PCR-Grade Water

Additional product information

Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.

Mutation detection kit tech note

Read about the Guide-it Mutation Detection Kit and see data comparing it with a standard CEL nuclease assay for detection of CRISPR/Cas9-induced mutations in mammalian cells.

See data Gene editing product finder

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The CRISPR/Cas9 system for targeted genome editing

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CRISPR/Cas9 genome editing tools

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SNP engineering application note

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Guide-it Mutation Detection Kit

Notice to purchaser

The Guide-it Mutation Detection Kit is used to confirm the presence of mutations in genomic DNA

Comparison of the Guide-it and Surveyor assays for detecting mutations in mammalian cells

Successful knockout of AcGFP1 in HT1080 cells using the CRISPR/Cas9 system

631448: Guide-it Mutation Detection Kit