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Wt= CCGC
Mut=tCag
GGCAAGACACTGGTGATGGCTGTGTATGATTTTGA(C/t)C(G/a)(C/g)TTCTCCAA
GCACGACATCATTGGAGAGTTCAAAGTTCCTATGAACACCGTGGATTTTGGCCAC
GTCACCGAGGAGTGGCG
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
19749 | GCT CCC TAG CAT CCT GTG AC | Forward | A | |||
19750 | CAA TGA TGT CGT GCT TGG AG | Reverse | A | |||
19751 | Fluorophore-1 | GTG TAT GAT TTT GAC CGC TTC TC | Quencher-1 | WT Probe | ||
19752 | Fluorophore-2 | TGT GTA TGA TTT TGA TCA GTT CTC C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
19749 | 0.40 uM |
19750 | 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 |