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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.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
14825 | AGT GGA TAC CAA TGC TGC AG | Common | A | |||
14826 | TGG TTT TTC CAG GGA GAC TG | Wild type Reverse | A | |||
14828 | GGG CAT TCA TAT TAC TTT ATA ATC CTC G | Mutant Reverse | A | |||
14829 | Fluorophore-1 | TCT GCA ACT GGT CTT CAA CCG TCC | Quencher-1 | MUT Probe | ||
14848 | Fluorophore-2 | ATG GAC GGT TGA AGA CAG GGC A | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
14825 | 0.40 uM |
14826 | 0.40 uM |
14828 | 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 | -- | repeat steps 2-3 for 40 cycles |