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amplicon = 85 bp
GACATGATGATAGTAGGTCAGACAATTTCAATACTGGAAACATGACTGTTCTTTCACCATATTTAAATACTACGGTATTGCCCAGCTCTTCTTCCTCAAGGGGAAGTTTAGACAGTTCTCGTTCTGAGAAAGACAGAAGTT(t/a)GGAGAGAGAGCGAGGTATTGGCCTCAGTGCTTACCATCCAACAACAGAAAATGCAGGAACCTCATCAAAACGAGGTCTGCAGATCACTACCACTGCAGCCCAGATAGCCAAAGTTATGGAAGAAGTATCAGCCATTCATACCTCCCAGGACGACA
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
13779 | GGG AAG TTT AGA CAG TTC TCG T | Forward | A | |||
13780 | TGT TGG ATG GTA AGC ACT GAG | Reverse | A | |||
13781 | Fluorophore-1 | AGA CAG AAG TTA GGA GAG AGA GC | Quencher-1 | MUT Probe | ||
13782 | Fluorophore-2 | AGA CAG AAG TTT GGA GAG AGA GC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
13779 | 0.40 uM |
13780 | 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 |