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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 104 bp
Wild Type = 133 bp
>chr1:127306714+127306846 133bp ATGAACTTGCCTTCCCCTTC ATGAAGCCGAAAGTCACCAG
Wt Sequence: ATGAACTTGCCTTCCCCTTCTGCTTGTTGAGAGCCAagGGAATGGTACATTGCTAGAGAGAGATGGCTTTCTTTTCTCCCTGGAAGTTGTCCTCTCAGAAGCTGGGCTTTTTCCTGGTGACTTTCGGCTTCAT
Mutant Sequence: ATGAACTTGCCTTCCCCTTCTGCTTGTTGAGAGCCAAAGCTTATCGAGCTTGCATGCCTGCAGGTCGGAGGCCACCATGGCTCGCGATGATCCCGTCGTTTTAC
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39432 | ATG AAC TTG CCT TCC CCT TC | Common | A | |||
39433 | ATG AAG CCG AAA GTC ACC AG | Wild type Reverse | A | |||
39434 | GTA AAA CGA CGG GAT CAT CG | Mutant Reverse | A | |||
39435 | Fluorophore-1 | TGA GAG CCA AGG GAA TGG TA | Quencher-1 | WT Probe | ||
39436 | Fluorophore-2 | TTG CAT GCC TGC AGG TC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39432 | 0.40 uM |
39433 | 0.40 uM |
39434 | 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 |