What oligos do I need to order to perform the assay?
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 the performance of the designed displacement and flap-probe oligos
Oligos used with the Guide-it SNP Screening Kit do not require special modifications such as fluorescent labels or quenchers, but the kit protocol does require the inclusion of a hexanediol (/3C6/) blocking modification on the 3' end of the flap-probe oligo. Inclusion of this modification is a standard option provided with oligo synthesis services, and no special purification step is needed in this case, only standard desalting. To simplify the oligo design process, we have developed an online software tool that outputs assay-specific oligo sequences in a ready-to-order format. Additional information regarding the design of the assay-specific oligos is provided in the Guide-it SNP Screening Kit User Manual.