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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 96 bp
Wild Type = 82 bp
>chr2:158614981+158615062 82bp TACCGAACCAACGCAGAGG GGGAATGACGGGGAGTGG
Wt Sequence: CTACCGAACCAACGCAGAGGACTAGGGGGTGGGGACCCTGCCCCCAGCTCCATCCCCGCCCACCCCCACTCCCCGTCATTCCCGCCCCCACCCCCACTCCCAGCCTCCTGCGCCGTCGCGCTAGGGAGGCCGAGCTTTAA
Mutant Sequence: CTACCGAACCAACGCAGAGGACTAGGGGaatTCCGCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGT
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
13007 | ACA CCG GCC TTA TTC CAA G | Mutant Reverse | A | |||
45508 | Fluorophore-1 | TAG GGG AAT TCC GCC | Quencher-1 | MUT Probe | ||
47594 | TAC CGA ACC AAC GCA GAG G | Common | A | |||
47595 | GGG AAT GAC GGG GAG TGG | Wild type Reverse | A | |||
47596 | Fluorophore-2 | CTG CCC CCA GCT CCA TCC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
13007 | 0.40 uM |
47594 | 0.40 uM |
47595 | 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 |