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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= 136 bp
Wt= 128 bp
Wt Sequence:
Mutant Sequence:
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
33265 | Fluorophore-1 | AAG CAA CAT GGC ATT ATT TCA A | Quencher-1 | WT Probe | ||
33266 | Fluorophore-2 | AGA AGC AAC TTG GAT GTG AGT G | Quencher-2 | MUT Probe | ||
33267 | TGT GGG TAA AGG TGA GTG CA | Common | A | |||
33268 | CAG GAC AGG TGT TTC GGT TT | Wild type Reverse | A | |||
33269 | TTC GAG TTT ATC GAG GCT GAG | Mutant Reverse | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
33267 | 0.40 uM |
33268 | 0.40 uM |
33269 | 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 |