Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mut = 91 bp
Wt = 94 bp
Fam = Mut
Hex = Wt
Wt Sequence:
gctctctggtgtggcatctgggctcgtcctggaggagggggtcgcctagaggaactgggaacagactgaggcagggaaggaggggggtggggcaggagaggcgccagctcaagttcagccacgataaaactgagggccctctgaacTCGAGGGGAGGCtcaggccgtcctctcttccttccatccgggggaatgtgctccagatacccacagccctcacgcaccgcacctccaaccaacccgtcccctccctaggaagaggagcgaaggcacgaggcaggcgaggggcggggagaggcgctgacaaatcagctgcgggggcgacgtgaaggagccagggagccagagcgcccggcagcaggcagcagacggcaggagaccagcaggtgttcccc
Mutant Sequence:
TCGCCTAGAGGAACTGGGAACAGACTGAGGCAGGGAAGGAGGGGGGTGGGGCAGGAGAGGCGCCAGCTCAAGTTCAGCCACGATAAAACTGAGGGCCCTCTGAACTCGAccgcatgcctgtacactccggaagacgcgttcggccgtgctagcgaattctggatccacgatctagagtcagcttctgatggaattagaacttggcaaaacaatactgagaatgaagtgtatgtggaacagaggctgctgatctcgttcttcaggctatgaaactgacacatttggaaaccacagtacttagaaccacaaagtgggaatcaagagaaaaacaatgatcccacgagagatctatagatctatagatcatgagtgggaggaatgagctggcccttaatttggttttgcttgtttaaattatgatatccaactatgaaacattatcataaagcaatagtaaagagccttcagtaaagagcaggcatttatctaatcccaccccacccccacccccgtagctccaatccttccattcaaaatgtaggtactctgttctcacccttcttaacaaagtatgacaggaaaaacttccattttagtggacatctttattgtttaatagatcatcaatttctgcagacttacagcggatcGATCCCCTCAGAAGAACTCGTCAAGAAGG
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52272 | CAG CCA CGA TAA AAC TGA GG | Common | A | |||
52273 | TGG GTA TCT GGA GCA CAT TC | Wild type Reverse | A | |||
52274 | GAT CCA GAA TTC GCT AGC AC | Mutant Reverse | A | |||
52275 | Fluorophore-1 | ACT CGA GGG GAG GCT CAG G | Quencher-1 | WT Probe | ||
52276 | Fluorophore-2 | CCT GTA CAC TCC GGA AGA CGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52272 | 0.40 uM |
52273 | 0.40 uM |
52274 | 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 |