Table 2 Primer sequences and reaction conditions for gel-free genotyping assays
Assay name | Target locus | Primer 1 | Conc. (nM) | Primer 2 | Conc. (nM) | Primer 3 | Conc. (nM) | Primer 4 | Conc. (nM) | Expected results |
|---|---|---|---|---|---|---|---|---|---|---|
Z/EG | Any transgene generated using the pCAGG construct (e.g., Z/EG line) | TCGA TGCA GGAT AACTT CGT | 400 | GGT ACC GTC GACT GCA GAAT | 400 | AGC AGC AGG CAG GGC TTT | 50 | GTCT GGA CAC GGG AGC ACTT | 50 | Primers 3 & 4 generate a single peak in all samples (control product, gdf); primers 1 & 2 generate an additional, lower Tm peak (transgene-specific product) in ZegGFP/0 or ZegGFP/GFP samples. |
Cre | Any transgene containing cre (e.g., Pdx1-cre line) | ACATT TGGG CCAG CTAAA CAT | 200 | CGG CATC AAC GTTT TCTT TT | 200 | GGC GAG AGC AGA GTGT GGA T | 200 | AAGT CGG CAG GCA CAG GAG | 200 | Primers 3 & 4 generate a single peak in all samples (control product, k17); primers 1 & 2 generate an additional, higher Tm peak (transgene-specific product) in any sample with a cre transgene. |
PdxCre | cre fused to 5' regulatory region of pdx1 gene (Pdx1-cre line) | TAAG GCCT GGCT TGTA GCTC | 200 | ACC GGT AATG CAG GCA AAT | 200 | AGC AGC AGG CAG GGC TTT | 30 | GTCT GGA CAC GGG AGC ACTT | 30 | Primers 3 & 4 generate a single peak in all samples (control product); primers 1 & 2 generate an additional, lower Tm peak (transgene-specific product) in pdx1-creCre/0 or pdx1-creCre/Cre samples. |
Rosa26 | Any rosa26 allele targeted using a standard targeting allele (e.g., Rosa26R line) | GCGC GCGC GCGT GATC TGCA ACTC CAGT CTTTC | 200 | GCG CGC GCG CGC GCG CGC GCC ACAC CAG GTTA GCC TTTA AGC | 200 | GAC AGG ATAA GTAT GAC ATCA TCAA GG | 200 | Â | Â | Primers 1 & 2 generate a single peak in samples containing the wild-type rosa26 allele; primers 2 & 3 generate a lower Tm peak (transgene-specific product) in samples containing a targeted allele; both peaks are observed in heterozygote samples. |