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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:102432318+102432411 94bp GGAAGGGCTCACCATGAAGT ACCGCCTACCCACAACTGTC
Mutant= 110 bp
Wild Type = 94 bp
Wt Sequence:
gggaagggctcaccatgaagttagctttaaggctgatccagtcTCTGTGTCCCCTTCCCTTGGgtggggtggggggacagttgtgggtaggcgg
Mutant Sequence:
agagggtgagctgcaatgttgctgagtctggtttccaactcctggggttcataacttcgtataatgtatgctatacGAAGTTATtaggtggatccacTAagaaggtgacagataaggatgccgaattaggggtctggggacacggagaggccatgctggagtggtttaacaccactggatgccttagttgtctctctgcagta
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
54745 | GGA AGG GCT CAC CAT GAA GT | Wild type Forward | A | |||
54746 | ACC GCC TAC CCA CAA CTG TC | Wild type Reverse | A | |||
54747 | AGC TGC AAT GTT GCT GAG T | Mutant Forward | A | |||
54748 | TCC TTA TCT GTC ACC TTC TTA GTG G | Mutant Reverse | A | |||
54749 | Fluorophore-1 | TCT GTG TCC CCT TCC CTT GG | Quencher-1 | WT Probe | ||
54750 | Fluorophore-2 | CTG GTT TCC AAC TCC TGG GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
54745 | 0.40 uM |
54746 | 0.40 uM |
54747 | 0.40 uM |
54748 | 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 |