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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 95 bp
Wild Type = 95 bp
>chr6:118692241-118692335 95bp GTCATCTTTGGATCCTTTTTCG CTCGGCTGCGTTGTAAAGAG
Wt Sequence:TCATCTTTGGATCCTTTTTCGTTCTAAATCTGGTTCTCGGTGTTTTGAGCgGgtaagctgaccacttctgtgtcctctttacaacgcagccgagcaaggtcccaggttcaatgcagaatc
Mutant Sequence: CTAACTCTCCTTCATCTTTCCAGATGCAAGACGCTATGGGCTATGAGTTGCCCTGGGTGTATTTTGTCAGTCTGGTCATCTTTGGATCCTTTTTCGTTCTAAATCTGGTTCTCGGTGTTTTGAGCaGGTAAGCTGACCACTTCTGTGTCCTCTTTACAACGCAGCCGAGCAAGGTCCCAGGTTCAATGCAGAATCCTCAGAGACAGG
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41979 | GTC ATC TTT GGA TCC TTT TTC G | Forward | A | |||
41980 | CTC GGC TGC GTT GTA AAG AG | Reverse | A | |||
41981 | Fluorophore-1 | TGT TTT GAG CAG GTA AGC TGA CCA | Quencher-1 | MUT Probe | ||
41982 | Fluorophore-2 | TGT TTT GAG CGG GTA AGC TGA CCA | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41979 | 0.40 uM |
41980 | 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 |