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Table 2 Primer sequences and reaction conditions for gel-free genotyping assays

From: Precise pattern of recombination in serotonergic and hypothalamic neurons in a Pdx1-cre transgenic mouse line

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    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.