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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= 106 bp
Wild Type = 96 bp
Wt Sequence: CAAGAAAAGCTGTGGCAACCGAAATGAGacTCCCTCAGATCCAGTGATAATTGACAGgtagggaccactcttctttcactggcgtcttagtcctca
Mutant Sequence: CAAGAAAAGCTGTGGCAAccGAAATGAGACTCCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAAC
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
24626 | GTT TAC GTC GCC GTC CAG | Mutant Reverse | A | |||
32921 | CAA GAA AAG CTG TGG CAA CC | Common | A | |||
32922 | TGA GGA CTA AGA CGC CAG TG | Wild type Reverse | A | |||
32923 | Fluorophore-1 | AGG AGC TGT TCA CCG GG | Quencher-1 | MUT Probe | ||
32924 | Fluorophore-2 | TGA CAG GTA GGG ACC ACT CTT C | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
24626 | 0.40 uM |
32921 | 0.40 uM |
32922 | 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 |