What do I need for the detection of the assay signal?

Detection of the assay signal requires only a standard fluorescence plate reader that enables excitation at 485 nm and capture at 535 nm. If the plate reader to be used has different possible filter sets, the Guide-it SNP Control Set provided in the kit can be used to determine the optimal combination of filters.Gene editing [...]

What is included in the Guide-it SNP Screening Kit?

The Guide-it SNP Screening Kit includes all necessary reagents for extraction and amplification of genomic DNA from mammalian cell samples, and for performing the Guide-it Flapase enzymatic assay: MightyPrep Reagent for DNA (Store at 4°C) MightyPrep Reagent for DNA Guide-it SNP Control Set (Store at –20°C) Guide-it SNP Positive Control Mix Guide-it SNP Negative Control Mix Guide-it Flap [...]

How does Guide-it Flapase detect the edited nucleotide?

Guide-it Flapase is a recombinant, structure-specific nuclease from Takara Bio; it recognizes and cleaves double-flap structures, but is unable to cleave gapped structures. The Guide-it SNP Screening Kit employs this feature to assay for single-nucleotide substitutions: the main difference between the double-flap and the gapped structures is whether or not hybridization occurs between the [...]

What oligos do I need to order to perform the assay?

Assaying a given nucleotide substitution with the Guide-it SNP Screening Kit requires the following oligos: -Forward and reverse primers (orange) for PCR amplification of the genomic region containing the target site -Displacement oligo (green) and flap-probe oligo (purple) for detecting the edited nucleotide in the PCR product, and SNP and wild-type (WT) control oligos (blue) for verifying [...]

What is the workflow for the Guide-it SNP Screening Kit?

The assay employed by the Guide-it SNP Screening Kit consists of the following steps: -Extract genomic DNA from clonal cell lines using MightyPrep (25 min) -PCR amplification of the target sequence (1hr 45 min) -Annealing of the PCR product and the oligo probles (25 min) -Perform the Guide-it Flapase enzymatic assay (1hr 15 min) -Fluorescence readout using a plate reader (5 min) As indicated, [...]