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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 87 bp
Wild Type = 95 bp
Mutation description: 1028 bp deletion
>chr3:8545128+8545222 95bp CTCTCAGCCTCCAGGTTTCT GAGGAAAGGGTGCATTTGG
Wt Sequence: agctgggcaaagcacagaaaggaggccgggaagcaacgtgcctccatgggctctgcttcactctcagcctccaggtttctgctgaagtctgagccctggcttccctcagtggTGGATTGTGTCGTGGAAGTGTAAGCCAAATGCACCCTTTCCTCCCAAGTTGGTTTTTGTCATGGTGTTTTGTCACAGTGA
Mutant Sequence: cttcactctcagcctccaggtttctgctgaagtctgagccctggcttccctcagtgGTggagggcagaggttggaaagcccagtaaagagcacttcccaagttccattccaagcatccatttcaag
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48340 | CTC TCA GCC TCC AGG TTT CT | Common | A | |||
48341 | GAG GAA AGG GTG CAT TTG G | Wild type Reverse | A | |||
48342 | Fluorophore-1 | TGG ATT GTG TCG TGG AAG TGT A | Quencher-1 | WT Probe | ||
48343 | TGC TCT TTA CTG GGC TTT CC | Mutant Reverse | A | |||
48344 | Fluorophore-2 | CCT CAG TGG TGG AGG GC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48340 | 0.40 uM |
48341 | 0.40 uM |
48343 | 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 |