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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= 96 bp
Wt= 120 bp
Wt Sequence:
Mutant Sequence:
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
34397 | ATA AAG ATA GGC ATT AAA CTC GCT TG | Mutant Forward | A | |||
34398 | Fluorophore-1 | TTG GTT AAT GGA CGT TTT GG | Quencher-1 | MUT Probe | ||
34399 | CTT AAC GCC TTT CCT TCA CC | Wild type Forward | A | |||
34400 | Fluorophore-2 | CAC CCC AAG GTC AAT GTG TC | Quencher-2 | WT Probe | ||
34403 | ATT CCT TTG CTC CTT TAG CTA TCC | Common | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
34397 | 0.40 uM |
34399 | 0.40 uM |
34403 | 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 |