Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.
>chr9:40317649-40317754 106bp ACGCCCCTTTCTTCACTTG ATAGCGGATGCTGGGGTAG
Mutant= 108 bp
Wild Type = 106 bp
Wt Sequence: acgcccctttcttcacttggcctcccagagACtatgggaagagagcttatctcgtcgtctttccaacacacaagcatccgccagcctctaccccagcatccgctat
Mutant Sequence: acgcccctttcttcacttggcctcccagagATggtagtagaaggttggaaatcaatgaaacagccactggcctttttaaaaatcctgccagtcgttggctgtcatact
474 bp deletion beginning at Chromosome 9 negative strand position 40,317,723 bp and ending after 40,317,250 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
36240 | ATA GCG GAT GCT GGG GTA G | Wild type Reverse | A | |||
36241 | ACG CCC CTT TCT TCA CTT G | Common | A | |||
36243 | AGT ATG ACA GCC AAC GAC TGG | Mutant Reverse | A | |||
36244 | Fluorophore-1 | TGA AAC AGC CAC TGG CCT | Quencher-1 | MUT Probe | ||
36245 | Fluorophore-2 | CGT CGT CTT TCC AAC ACA CA | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
36240 | 0.40 uM |
36241 | 0.40 uM |
36243 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | |
4 | -- | repeat steps 2-3 for 40 cycles | |
5 | 40.0 | -- | Forever |