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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 86 bp
Wild Type = 80 bp
>chr18:78857841-78857920 80bp TGTCCCCTGTGAGTGAGTCC GGAGGTGCTGTTGTTGTCTG
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48580 | TGT CCC CTG TGA GTG AGT CC | Common | A | |||
48581 | GGA GGT GCT GTT GTT GTC TG | Wild type Reverse | A | |||
48585 | Fluorophore-1 | AGC GAC AGT GGC ATC GG | Quencher-1 | WT Probe | ||
48778 | CGG TTC TTT GTG CTG GTG TC | Mutant Reverse | A | |||
48779 | Fluorophore-2 | AGA CTA TCC CTT GAC AAC CCA GA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48580 | 0.40 uM |
48581 | 0.40 uM |
48778 | 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 |