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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.
Mut= 110 bp
Wt= 113 bp
Wt Sequence:
Mutant Sequence:
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
34583 | CAA TGC CCA GAG CAG CTT AC | Common | A | |||
34584 | Fluorophore-1 | TAA GTG CAG GGG GTT GAA AG | Quencher-1 | WT Probe | ||
34585 | CAA TTC TGG TAC CTG ACT GAC C | Wild type Reverse | A | |||
34586 | CAC CGT GAC AAC GAG CTG | Mutant Reverse | A | |||
34587 | Fluorophore-2 | CTT ATG GTA CTT TAT GCT GTG TGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
34583 | 0.40 uM |
34585 | 0.40 uM |
34586 | 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 |