Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr18:37400953+37401101 149bp TTATGAGCAACACGGTGAAGA CATTACTTGCCTGTTTAGGTGA
Mut = 140 bp
Wt = 149 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TTTGACACTAAAGGGTTGTTTGCCTTAAAGCTAGAATTGTATCAATTATCCCAAAAAGACACAGGGTGGCGCTGCAGGCTAAGATTGTGATTAAAAAATCACTAAAATACCCGGAATCACTGTTGCAATACAGAACAAAGGAAGCAGACAAGAGCTTTGCAAGCACTATGGAGCTATAGAGGGTTTGGGGCAAGAATATAATGGATTAATATGAGATTATGAGCAACACGGTGAAGATTCCTCCAAAgagggaatcagaattcagtgtttcaaaacatttggagactgctacaggattggatgcaagcatgcatttcctcatcatggagaagctggggagaattcacctaaacaggcaagtaatggcatttatttttatgatggttttggttcaggtttgcagtgagccaacaattcgatactctatcttagaggaaacagaaagtggctcctttgtagcccacctggccaaggatctgggtctgggagctagggagctggctgccaggtctgcacgggtgttgtctgatgattacaagcagagattactgctggatcctgagactggagatttgcttctgagggagaaagtagaccgggaggaggtgtgtagcactgtggatccctgtgtgctgcatttccaggtgactcttgaaaagccagtgcaatattttcaaagagaattattgatccaggacataaatgatcatgctccagaattcccagatagagaactgctcttgagaataccagagaacagccagcaaggcacacaattttcactgaatttagcccaggacttggatgtgggcagcaatgggcttcaacaatacacagtcagccccaacccttattttcacgtcctcactcaaaataatagtaaaggcaaaaaatacccagagttggtgcaggacagaggcctggacagagaggagcaggcagagctgagcttaaccctcacagctctggatggcgggtctccaccgaggtcaggaacagccctggttcgaatcctgatcatggatatcaatgacaatgctcctgagtttgtgaacagtccctatgaggtgcaggtcctagagagcagtcctccagactccccagtcttgactgtcttagcacgggatgcagatgctggcaactttgggagagtttcctatggcttctttcaagcatcagatgaaatccaacaaacattctcaataaacgcaactagtggagacatgcgattgaaaaagaaattggattttgaaaaaattaaatcttaccatgtggaaatcgaggccatagatggaggtgggctttctgggaaaggctctgtaacgatagaggtggtggatgtgaatgacaatgccccagagctgaccatatcttcactgaccagctctgtcccagaaaatgctcctgagacgatcataagcatcttcagagttggggacagggattccggagagaatggaaagatggtttgttctattccagagaatctgccatttattctgaaatccaccttcaagaacttctacaccctggtgacagagagccctctggacagagaaagcagagctgagtacaatatcaccatcatggtctctgacatgggcacacccaggctcacaacatggcacaccataaaggtgcaggtgtccgacatcaacgacaacacccccgccttcacccaaacctcctacaccatgtttgtccgcgagaacaacagccctgccctgcacataggcaccatcagtgccacagactcagactcaggctccaatgcccacatcacctactcgctgctgccgccccatgatccggaactggccctctcttcgttaatctcgattaacgctgacaatgggcagctgtttgcactcagggcgctggactatgaggccctgcaggtcttcgagttccacgtgggcgccacagatggaggctcacctccacttagcagccaggctctggtgcgcgtggtggtgctggatgacaatgacaatgcgcccttcgtgctctaccctatgcagaacgcctcggctcccttcactgagctgctgcctagagctgcggagccaggctacctggtcaccaaggtggttgcagtggaccgcgactctggccagaatgcctggctatccttccagttgctcaaggccacggaacctgggctgttcagcgtgtgggcgcacaatggagaggtgcgcaccaccaggttgttgagtgagcgcgatgtgcccaagcacaggctactgctggtggtcaaggacaatggagagcctcagcgctctgccagtgtcactctgcaggtgctgttagtggatggcttctcacaatcttacttacctcttccagaggtagcgcgggatcccgcccatgaagatgaggacgtgcttacattgtacctggtcattgccttggcatctgtgtcttctctcttcctcctgtctgtgctgctgtttgtgggggtaagactgtgcagaagggccagagaggtctctctgggtggctgctctatgcctggtgaacactttcctggccacctggtcgatgtcagtggagcagggaccctgtcccaaagctaccagtatgaggtgtgtctgaggggcgactctgggactggtgagttcaagttcctgaaacccatgatccccaatgctgggatagaaattatggaaagtccccactGCAGGGATAGCTTTGTATTCAACTAAGTAATGAATCTTAAGAATTAATACACTTTTATATGGTTAAGTGTAACATTAACTAACTTTAGGAGAAACTACCTTGTGCATGTCTCCAACCGCTTTTGTTTATCTTATTCATATTACGCACGCTTAGTCT
This mutation is a 2432 bp deletion beginning at Chromosome 18 position 37,400,984 bp and ending after 37,403,415 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48946 | GAC ATG CAC AAG GTA GTT TCT CC | Mutant Reverse | A | |||
48947 | Fluorophore-1 | ACA TTT GGA GAC TGC TAC AGG ATT | Quencher-1 | WT Probe | ||
50580 | TTA TGA GCA ACA CGG TGA AGA | Common | A | |||
50581 | CAT TAC TTG CCT GTT TAG GTG A | Wild type Reverse | A | |||
50582 | Fluorophore-2 | TCC AAA GCA GGG ATA GCT TTG TA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48946 | 0.40 uM |
50580 | 0.40 uM |
50581 | 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 |