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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= 92 bp
Wild Type = 101 bp
Wt Sequence: ctatgtagcccaggctgtcctcaaagtcacactcacagagatcctcctgcctctgactccCGaattctgggattaaacagcggttgggattagactgcgtc
Mutant Sequence: CTAAAGCGCATGCTCCAGACTGCCTTGGGAAAAGCGCCTCCCCTACCCGGTAgaattctgggattaaacagcggttgggattagactgcgtc
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
33215 | CTA TGT AGC CCA GGC TGT CC | Wild type Forward | A | |||
33216 | GAC GCA GTC TAA TCC CAA CC | Common | A | |||
33217 | Fluorophore-1 | CGC CTC CCC TAC CCG | Quencher-1 | MUT Probe | ||
33218 | Fluorophore-2 | ACA GAG ATC CTC CTG CCT CTG | Quencher-2 | WT Probe | ||
oIMR5316 | CTA AAG CGC ATG CTC CAG AC | Mutant Forward | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
33215 | 0.40 uM |
33216 | 0.40 uM |
oIMR5316 | 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 |