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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= 105 bp
Wild Type =116 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
31552 | TGA AGA TAC TGT CCC ATA TAG AAG C | Common | A | |||
31553 | CCT GAG CAG TGG AGG AAG G | Wild type Forward | A | |||
31554 | AAC TTT CTG GGT ATG CAT GAT T | Mutant Forward | A | |||
31555 | Fluorophore-1 | TCC TCA CAG CTG TTG CTA TGT T | Quencher-1 | WT Probe | ||
31556 | Fluorophore-2 | CAA GCT GTA ACC TCC TGA GCA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
31552 | 0.40 uM |
31553 | 0.40 uM |
31554 | 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 |