Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 116 bp
Wild Type = 126 bp
Wt Sequence (deletions in lower case):
AGCTCCGGCCTGGGTTTTCTGTCCAAATCAGACCCTTGATGTCCAGACTCTTAAGGCTAGCCTTTGTATTTCAAGACATAGTTGACAAGATCATCACGATATAAGGACTTTATATCATGTGGTGTCATGGAATAATTTCCATTGAAAAGTTGTGTGTTGTGTTGCTGTGGACCCTGACCCTCCattttctgtaaggcgggcatagttgcacctccttcgtatcagttgtaaatgagacagctggagtaggacagatgcctgagcttgacacagagaccagggcatatggcaactgggaggcctgctttgtgtgtgcagtggcctctgttcacacagagtttcccctggcctccagggcaggagctctacctgctggagccctgtgcatgtctgtctgctggagagcagaagtggggaacccatttggcttcaaagattttttttcccctgttttaagggtgtcagcattgaagccatgaatgagagtaaaatggcgtcctcggagttaagctcggggcctgtggaaaaagctgccaagccgttgccatttaaggatcccaactttgtggtaagcatatgaggtggtatatcaatacagggtgctgtaccttagcttctgatctagctgggcctgaggtggctctggagtaccctcttcctcagtcccatttgccaccttctcctgtgggctgttggagacggtagtcgggggttgggagtcgggggttggggggagagagtggcagttggcggaaaagggatttgggactggacaggaaaggccctttgtgatctcccagtgtagcagaactttgaggaggcatcttttccatgattgtctccctgtgcttttttactggctttaaataattttaaaagatgcttgaaatatacaactaaaataggagtagtcctcataaccctttatagccctttgattcatgaaggttatattaagtagccagttacaaattataactttaaaggtttattgtttttattaatgagttttggcaatgtactgtagccagggtctgcaaatatgttagtagtcttttccacttcatgcttttaatctttatagcacagaaagaaatttccagaaggcttgccacaccatgattgaagcttatttgagctttccaggaccactggtgattatgaggctcagtgacacacccttcgagggggtttagagatgtagtctcgagattaaatccaactgtgtggtaaacttattttattttattttttttattttttatttttttttggtttttcgagacagggtttctctgtatagccctggctgtcctggaactcactttgtagaccaggctggcctcgaactcagaaatccacctgcctctgcctcccaagtgctgggattaaaggcgtgcgccaccacgcccggccaacttattttaaaggaatgagatcataagtgaattcaactagattggatcacataaagaatttttcctgtgtgcatgctgaaaaatagcatttacttctgtcccatcaccagcaaacttgtgggtttgaagagcccccaaactaggtcctttttgtttgcttgtttattttcatcttttgttttttgttttttgataaaggtcacctgtagcccaggctggcctcaaactccctactcttctgcttctacctcctgagtgctggcattgtagtgacatccaccacgcatggctaaaaactatgttctttcaaacaaagatttaagtgtaggtgaggtacagatagttactgatggaccgagaggtgcctgacaaccctaaagggcaggggcagggcgtcacgcttctccaggcgcgtcttccactgacttctcttcaaaccttcctttaattcaagcactctggccatggcggcgctgtagctggcaagaagaacagaacctggaagaacctcaaacagatcctcgctgctgaaagggcgttgccatggcaactgaacgatcctaactgtgagctgtctagagccttccatgtgtttgtaatgcagacaggaacttccttacaacaagcctacgttatttgctcgtgtttgtgttacttcagggaaatattcttagcctgagacccgcacagtggcacccatccagggatagtctgcatgtattccctttccactgagttggtgttgaggctgtttgtttgtcacggctgtgtgggttttagtgtcattatttaaaatttcagtctggagaaataggtcccgccgagataatgttgatgcttctattaagcttcccattttaaagtctactctttaagtttgttttcacaaacaacgtagcccacctaatgtgagaactggaaagtctccagcgtgcaggtaccagccactctctcactctctcctcccactgtagcatgtcctggcctgggcagaatccgttggtctactcaagtgcattggaaactttaggttccaagacctctccacctcctgccccgctgttcgtctttcatgggggtgactcagagggtgacaagttagtcatctgggtgtggactgcttgagtctgggtgtgtggatccagctgatggctgtgtggcctttcttcattgcctgttccactcagctggagcctgtgaagtagcctgcgggtagatgaacctggccgttccactggaggaacgtgtctctggtagcaaggcaggaccccagctgaggcccacacttagacatgctgggctggcttttgagccgtctctttttaagtgtgtaatcacaaaggaatatttcaggactgctgtcatgtgttagcttagagtaaaggagggggagtcgggatagttagaaggttatttggtgatcattttcttttctttcttttagactttagtattgatgctcctccatccttcaagccagctaagaagtattctgatatttcaggccttcttgtgagtataattacatctgtctatattagcactaaatagatttaagttatgtctctctagACAGATGGCTCTGCCTGGAGTCCATGCTGTGACCTGAGTGTCTGTGTGGGTAACCTGGAGGGGCCATTGCTCCAGTAAGCAAGACACAGAATTTTTACTTTTTTCCCTGTGATAGTTAACTGTATGCGACAACATGAATGAACCACCTTTCTGTTTTACTCTTGAGGTTTTAAAATACTCATATACAATTGGTGGTAGTTTAAAAACAAAT
This mutation is a 2807 bp deletion beginning at Chromosome 18 position 24,111,655 bp and ending after 24,114,461 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48303 | CCT CCC ACT GTA GCA TGT CC | Wild type Forward | A | |||
48304 | CTG AGT CAC CCC CAT GAA AG | Wild type Reverse | A | |||
48305 | TGT GTG TTG TGT TGC TGT GG | Mutant Forward | A | |||
48306 | CTT GCT TAC TGG AGC AAT GG | Mutant Reverse | A | |||
48307 | Fluorophore-1 | CAA GTG CAT TGG AAA CTT TAG GTT | Quencher-1 | WT Probe | ||
48308 | Fluorophore-2 | TGA CCC TCC ACA GAT GGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48303 | 0.40 uM |
48304 | 0.40 uM |
48305 | 0.40 uM |
48306 | 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 |