Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Wt Sequence (deletion in lower case):
ATGACAGATGGTTTTACTTTATAAATCAGTCTTCTGATTCTGTGTAGAATATAGCGTGACTTACATTATGTTTAAATGGATGCCCTGAAGAAAGCTGTGAGCCACTCTGTTATGATGGTCTAGCAGACAACCTGTGGGGAAGCAGAGGGTGGGGAAGCAGAGggtgcttgcctcggcataaagtgagcaaggtggacttgctcttctatagacccgtagcttccctgcctcgtgctggtttctcctgactgacttactcacaccaagtatctatcttttccagcctctagcccctgggtctggcacgatcaacatggagaaacctcaaaggtacccatcctggtacacagctagcagagacacgccacacactccctctgctaactccttgcttgattgatgggggacaccaaggaccagagagttcaaagtctttgcttaagggttgggggacagcagtgaccagaaccacttcctgccccctcatcctcccagaacccatgacacccgtgactcctctgggcgaatgagagagaagtgggaaagaccagagagatggaatccctgtgactgcacagggcctcctccctctgctcctgggggccttgcagggtttggactggagtcattttaccctggactcagtgtcctcctcagctttgggcaggactagatagcttgggaagaagatcctgaggaaactgtttatccttcactacccccagagcaagtataagccaaagcttgttatgaaatggcccacgatggccaaggaaaaggctcctgtgcacaacaaacgtagtctgatccttgtgtgctcgcttcttacagcatcaagaaccaaccacaggatgtatgcatgggaatcatgacaggtgagtcctgggtttccagccagactccaaatcgtccccaccccctgggacacagcagatgttttgttaataaagaagatgcactttattgcttcattgcatcactagtgactgggaagacgggaaggcatgctacggagacataagGGAGGCAGGGGATAGAAGACGTGTGACCACACGTCCTGAATAGAAAGCATGAGAAACATTCAGGGTAAGGGGGAACTGGAAGGACCAGACCAAAGCCTGTAAGATCTTGCTCCTGTATGACTACCTCAGAGGTGGATTCATGGCACTTACCTGAGAAAAATG
Mutation details
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47032 | ACG AGG CAG GGA AGC TAC G | Wild type Reverse | A | Wt R | ||
47034 | Fluorophore-1 | AGC AAG GTG GAC TTG CTC TTC | Quencher-1 | Wild type Reverse | ||
49020 | CAG AGG GTG GGG AAG CAG | Common | A | Common | ||
49021 | ACC CTG AAT GTT TCT CAT GCT | Mutant Reverse | A | Mut R | ||
49022 | Fluorophore-2 | AGA AGA CGT GTG ACC ACA CGT CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47032 | 0.40 uM |
49020 | 0.40 uM |
49021 | 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 |