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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:87862393+87862528 136bp GGATTCTAAGCTCCTTGGC TCCCGCGTTAGTCTCGTGTA
Mutant= 109 bp
Wild Type = 136 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44089 | TCC CGC GTT AGT CTC GTG TA | Common | A | |||
44091 | CCT AGA ATT GAT CCT GGC GTA | Mutant Forward | A | |||
45529 | Fluorophore-1 | ATA GCG AAG AGG CCC GCA C | Quencher-1 | MUT Probe | ||
69186 | GGA TTC TAA GCT CCT TGG C | Wild type Forward | A | |||
70096 | Fluorophore-2 | CTG GGA CCT TGG AAC ATT G | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44089 | 0.40 uM |
44091 | 0.40 uM |
69186 | 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 |