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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 115 bp
Wild Type = 110 bp
>chr7:110852071+110852180 110bp GAACTCTGCAGTCTTTTTCTGC GAAGTTTAGATGCAAGAGAGTGG
Wt Sequence: GAACTCTGCAGTCTTTTTCTGCAGGAACTGACAGTGGCTTGCTTCTTTGGTTCCCGGGGCATGAAACTTGCCTCGTGTGCACCTTCTCCACTCTCTTGCATCTAAACTTC
Mutant Sequence: GAACTCTGCAGTCTTTTTCTGCAGGAACTGACAGTGGcgGTGGCGGCCGTCTGTTAATTAAGGTGTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATTTGGTACCGG
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45741 | GAA CTC TGC AGT CTT TTT CTG C | Common | A | |||
45742 | CCG GTA CCA AAT TCC GAA G | Mutant Reverse | A | |||
45743 | GAA GTT TAG ATG CAA GAG AGT GG | Wild type Reverse | A | |||
45744 | Fluorophore-1 | CGG CCG TCT GTT AAT TAA GGT | Quencher-1 | MUT Probe | ||
45745 | Fluorophore-2 | CTG ACA GTG GCT TGC TTC TTT | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45741 | 0.40 uM |
45742 | 0.40 uM |
45743 | 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 |