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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.
Mutant= 118 bp
Wild Type = 117 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
31570 | GCT ATG GAA CAT GCT GGA AA | Common | A | |||
31571 | AGG CCA GAC CCA GGA GAT A | Wild type Reverse | A | |||
31572 | CCA AAG GTT CTG ATG TGT GTC | Mutant Reverse | A | |||
31573 | Fluorophore-1 | AAC ACC TCC CTT CCT CTT TGA | Quencher-1 | WT Probe | ||
31574 | Fluorophore-2 | TGT AAT GGT GAA GGC ACA GC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
31570 | 0.40 uM |
31571 | 0.40 uM |
31572 | 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 |