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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 85 bp
Wild Type = 110 bp
>chr9:119961380-119961489 110bp CGTGATCTGGTTTCTCCTTCC CCAAATAACAGGCCTCAGCA
Wt Sequence: tcctttaaggctcacgtgatctggtttctccttcccctccagGACCTCACCATGTCCACCTCCTTCCCTGAACTGGATCTAGAGAATTTTGAGTATGACGATTCTGCTGAGGCCTGTTATTTGGGCGACATTGTGGCCTTTGGAACCATCTTCCTGTCCGTCTTCTA
Mutant Sequence: GGACCTGCTTACTGCATGCAGCCAGTGAGAACTACAATCCTTTAAGGCTCACGTGATCTGGTTTCTCCTTCCCCTCCAGGACCTCccgcgggcccgggatccaccggtcgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgac
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
31017 | CGT GAT CTG GTT TCT CCT TCC | Common | A | |||
32856 | CTC CTC GCC CTT GCT CAC | Mutant Reverse | A | EGFP | ||
39210 | CCA AAT AAC AGG CCT CAG CA | Wild type Reverse | A | |||
39211 | Fluorophore-1 | CCA GGA CCT CCC GCG GG | Quencher-1 | MUT Probe | ||
39212 | Fluorophore-2 | CCA GGA CCT CAC CAT GTC CAC C | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
31017 | 0.40 uM |
32856 | 0.40 uM |
39210 | 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 |