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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.
>chr4:147810905-147811012 108bp GAACCTTTTGCATGTTCTTGG CGCCAAGTTTGCCTTTTAGG
Mutant= 93 bp
Wild Type = 108 bp
Wt Sequence:gaaccttttgcatgttcttggctcctctcATctctgtgtacccggagctctgggcactgtgtggctgaaacttttttttgggacaaatcctaaaaggcaaacttggcg
Mutant Sequence: gaaccttttgcatgttcttggctcctctcAatatggtagaggtcacctcattaccagtgatgaatttgggaacagtttgaccagggttgatgg
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
33590 | GAA CCT TTT GCA TGT TCT TGG | Common | A | |||
33591 | CGC CAA GTT TGC CTT TTA GG | Wild type Reverse | A | |||
33592 | CCA TCA ACC CTG GTC AAA CT | Mutant Reverse | A | |||
33593 | Fluorophore-1 | CAC TGT GTG GCT GAA ACT TTT T | Quencher-1 | WT Probe | ||
33594 | Fluorophore-2 | TCT TGG CTC CTC TCA ATA TGG T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
33590 | 0.40 uM |
33591 | 0.40 uM |
33592 | 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 |