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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 109 bp
Wild Type = 122 bp
>chr11:102432793+102432914 122bp CACACTGGTAGGACGCTGA TCTACTCTGAGGCCACGCACT
Wt Sequence: gccagctgacgcatgtgcgcttgctgcacactggtaggacgctgacacaagcgacacatctgcttgctttcctgttggcttgttgagaagactcaatgaagccagggccttggtcagagcacaggggagtgcgtggcctcagagtagaaggtggcacagagtttaaggtggctgccaaactttgcttggcatttgtagGCAGACCATGTGGGTCCTGA
Mutant Sequence: TGACGCATGTGCGCTTGCTGCACACTGGTAGGACGCTGACgtcgacATAACTTCGTATAGCATACATTATACGAAGTTatattaagggttccggatcaacgaagttcctattccgaagttcctattctctagaaagtata
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40717 | GAG AAT AGG AAC TTC GGA ATA GG | Mutant Reverse | A | |||
47089 | CAC ACT GGT AGG ACG CTG A | Common | A | |||
47090 | Fluorophore-1 | AAG GGT TCC GGA TCA ACG AAG T | Quencher-1 | MUT Probe | ||
47091 | TCT ACT CTG AGG CCA CGC ACT | Wild type Reverse | A | |||
47092 | Fluorophore-2 | AGA CTC AAT GAA GCC AGG GCC T | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40717 | 0.40 uM |
47089 | 0.40 uM |
47091 | 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 |