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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= 126 bp
Wild Type = 128 bp
Mutation is a two bp deletion (aa)
Wt Sequence: TGTGCAGTGACATCTTCCTTCCTAACTCCTCTCCACGCaaAGTGCAGCGCAGCAGCTGCGATCCAGGCGGGGCTGGGTGCTGGGGAAGCTGACGCGGAGGCGAAActgacacacagacatcgttgcag
Mutant Sequence: TGTGCAGTGACATCTTCCTTCCTAACTCCTCTCCACGCAGTGCAGCGCAGCAGCTGCGATCCAGGCGGGGCTGGGTGCTGGGGAAGCTGACGCGGAGGCGAAActgacacacagacatcgttgcag
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
31097 | CGA GCA GTG ACA TCT TCC TTC | Forward | A | |||
31098 | CTG CAA CGA TGT CTG TGT GTC | Reverse | A | |||
31099 | Fluorophore-1 | TCT CCA CGC AAA GTG CAG | Quencher-1 | WT Probe | ||
31100 | Fluorophore-2 | TCT CCA CGC AGT GCA GC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
31097 | 0.40 uM |
31098 | 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 |