Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:19525099+19525241 143bp GCTATCTGACCCACCTACTTCC GCGTCTGCCCTTCATCATC
Mut= 141 bp
Wt= 143 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CATTCATCTGTTTCTCTATCCATTAATATTCTTGTACCGGGTCATTTCATTCATTCCTGGAGTGGCCACAACTTAAGGCCCTGTCTTTCTTGTGACATCCTGGCTGCCTCACCCTCTTGCCTGGTCCTGGCTCTTTCTACCTCCCTCCCAtcctttctcagggaggtccctacttttgtcctagacctccaggcctcttccttcccacctacatgcaccaccagcctctccccgacccgtctccactgacgccttgcctcctctcccccagcgctgatgcagcaggaggccgcgcagcgtgagagcgaggagctgcagcgcgtgcagtggcagccacgtcgcgtgcgcggctggggcgtcccgaagctgctgtggttcctggtgtttctgcagcccgtgatcacggagctgcacctgcggcgcaggaatgtgcgcttcctgttcatccgcttcagcgcctggcagtacgcgggcaccgacaagctgtgggccgggctggtgaccacgctgtgtgagggcatccgccaccactacggcgcgctgccctttagcgtgtactcggtgctaggcaacaagccctgtggcccgcgtgatggcctctgccaacgcgagtggcactgccgacgtcgcgtgtgcctggcgctgctggcgctgctggccgcgctgtgcctgggtgtggggctgctgtacctgtcgctgggaggccacgcgccgggccacggagagcgtggtgtgctcaaagcgctgggcggcgcggccaccacgctatccggctcagggctgctcatggccgtgtactccgtgggcaagcatctgttcgtgagccagcgcaagaagatcgagcgcctggtgtcgcgtgagaagtttggcagccagttgggcttcatgtgcgaggtgaagaaggaggtggaactgctcacggatttcctgtgcttcctggagatctaccagcgacgccggctgcgtgtggtcctcgaggtcactgggttggacacgtgctacccagagcgcgtggtgggcgtcctaaacgccatcaacactctgctgtccgacagccacgctcccttcatcttcatcctggtggtggaccccagcattctggccgcgtgcctggagagcgcgggtaacatgaagggcacagcagacaacggctacctgttcctcaaccgcacagtcacgctacccttctccgtgccgattatgggccgtcgcaccaagttgcagttcctgcacgatgcggtgcgtagccgggacgatctgctgttccgggaattgacgatcaagctacagccgcagagccccgggaacttgggcgcaggtgagggtactcagctgctggcggtggagacgcagggggacgccgagcgcacacagggccgcgtggacgcggaggcggcgcggcgtatccaggaggcgctgtgctgcctgcatgacgagggtgattgcctgtatgagtacgtgcccgacaacgtggtgtccatgcggcgcattgtcaacacggtgcccatcaccgtgcgcctgttgcagcagcagcagcagcaacagcccgaccgcgtgggacccacaccgcgccacgcggtggcctgggtcgtcctcgctaaccagtggccttgtcgcctcagctgggtgctgcaatgcctggaggaccggcagcaggcagggggcgcgccagaggggcgctcgcgtctctgggatgtcttctgcgacaatagtcgcgagctgcacaccatgaccaaggccttgcagaacgtactggacctggatggcgaccctgagctttttgagcgctttctaggcactgatttccccttcaccgtggccgaggcgcagagtttgctgcgctgcacagtcaacctggaccattccatccgccgccgcatgggcctcatcagggccgtcagcgcgctcaagccgcccagcccacccaagtccccatcccaggacggtccccaggccagtcccagagccattatcgccgcaggcacgtcccatgcgggccaaggatcaggacacagtaaagaggcacatcagacccgggaccggacccatgggggcaagccaaggccgatggcctaagtccttaaatttcaggagcgtagaggctgtttgggtgtgggatggagaacaccagccacgggagacagcaacatccctcctgacctgatgagggtgtgagggcaagaggggctcaaaaccaatggggtatggccacacaccctgatgactttggaagccaaggggaagcaggtactactagcttcctggagtaggcagcttctacaccagtgtctgagtgagcagaggagtcagtgaggaacacagagtcaaagaagcaataaaggaagctgtggaaagccagggctgtctttcatgtcctggatttctggctaggtgagaatgctatctgacccacctacttcctcctcagggagccaggcctctggactgcaccctggcagctgaagttggcatcccAGGTGGTAGGAGGCTTGAGGGGGTGGTGGGTGAGGCTGCGGGGGCAGGGATGATGAAGGGCAGACGCCAAGTTGACTCAGGCAAAGGGGAGCACCCAGGCCTCAGGGGATGCCAGGCCTTTGTCCCCACACCCTAGCCTGCCCTCTGTGGGCAAAGCATGAACTGGCAC
This mutation is a 2325 bp deletion beginning at Chromosome 7 position 19,522,850 bp and ending after 19,525,174 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46192 | GCT ATC TGA CCC ACC TAC TTC C | Wild type Forward | A | |||
46193 | GCG TCT GCC CTT CAT CAT C | Common | A | |||
46194 | GGC CCT GTC TTT CTT GTG AC | Mutant Forward | A | |||
46195 | Fluorophore-1 | CAG GGA GCC AGG CCT CT | Quencher-1 | WT Probe | ||
46196 | Fluorophore-2 | CCT CCC TCC CAA GGT GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46192 | 0.40 uM |
46193 | 0.40 uM |
46194 | 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 |