Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:126585346+126585475 130bp GTGCCTTCACTCTTCTTTCTTC CCTCAGTCACCTCTCCCAGT
Mut= 136 bp
Wt= 130 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GTACCCACTTATTCAAGCTAGGCCTTGTCGGGGTCCCTTAGCAGACGTCCAGATCCCTGATTCTTACCTGGGCTTTGTTAACAGTGTACTCCTGGGGAAGTGGTTAGACCTCCCCTGAGTGCCTTCACTCTTCTTTCTTCTCTTCTAGGACTCcttcagtgcgtttgtgtcctacctcgtaggagacacagtccccacagtagagaggcagacacaggcagctgaggaactgggagaggtgactgaggggaaggttgtaggagaggaagcccaggaggtactggagggacttagaagtggccagagtgagggggtagaagcacccgaagagactagaagatgccaagaaggaagtttagctggtgaacagacctgggggtggagagcagatagctctgcaaggccccaagcagaaaggcaggacactgggtcctggaaggcagctgaggatgccaggggccaggagccaagtgtcccactgaagcctgaggcagagcctgggactcacagagacaggagcagtaatacagcccaggagatctgggagcacggtgaagaggaagcaagcagtggggagcctctgagaacctgtgaacagaaggaagaagaggaggaggtggtcagagcagcagagtcagggatggccgagggggtggagtcacagccaacctggcacagcgagcctggggggaatgctggcactgaggggcagcatgtgacagaggacagcaaagagatagactgggtggccaaagacatggttgcagaaatcgaatggtttggtgccaaagggattgacaaggaagaggagaggatggtcccaatgagggatggtgaaagggcaagggcacaggggacacaatgccccggggccgagtctgaggaccaggccatgttgagtagagaggcctggacagtctcagacagggagggggctgatagcctaggagttcagggaacagaatacgggtcagacccaggagacaacttcccaggaactactgggagagtctgggtcctagaagaggctgacaagggagaccagcaggatgaggtcgacgagaagagagaggctgaagttaggtttccgatccagaccctggaggctgagagaactggagagatgactgaaggccacatagctgaagaggaggctatgggagaacaggaaacagagggcagctttgaggatgaggagaggcaagacttagccatcagggataatggagtgagtctagaagaagaggtacgggcagaagaatcctccagggagaaaaggaactcctgggccacagagcctacgctagtcctagacacggaggctaaagatgaacctgactgggaagactctccagaagtcagcactgaggagttgtttgtgggagagaggagtgaagccgcccagatgacaccagaggtattgagagtaaaggtcactgaaggacaggaccctgagctggtgagacattcccaggctttaactaaacaacttgaggaaggacagaagggtcaggaagagactagtggagctccagacctgagcccagagcgggtgctaagcttgaaggaatatcctgggcctgtggggtttgcaggccctgagctagaagcctggggaaactggagtaggggtgtggaccgcagaaatagccaggaggtaaaagcagatgctgaagcaggcaaggaacagactgcaaccgagcaggcagtggagatccgggctgaaggaggccaggaggcccaacagccagaggtcttcgggtcaggaggagaggaggcactgacctccgtagcacttaacccggagctggagggaagccaaggagcagaggcggggacagaagagtcagtggaggaatccaagcccacagaaaacgaggctgctgaggaggaggctgtagtgccttgggaggcagatggaacttgcaggaagaggaggctggaggaggtgactctgagcctgcaagacagtgaggacacagagaccagttatttggctgaggagattattgtgggtatcagggccgtggataccgaggaagggccaaaatgggaagctgggttggctccagaaacagagcttgggaaagcctggtgttccgagggcagaggggaggctgggagaggcacggagctggaggagactacagagaagcaaagtggacaggaagttggcttggtgggctcagcagagaaggtgtctggctatgatatccaagaaattgatgggactgaagagggggagcaggcagagatggagacatctgtaatggcagaagacataagggggacggatggtgtgactttaggctcccaggcagagagagcagaggggtccataacccccatggagactgaggggctcctaagagatcagatgctgttggaagaagaggctgggggagggcagtcgcgagagcagaaggtccacaactcagagggcgagatccaaacgctggacgactcttcagaccaggaggggcagcagacacaccaaatccccacagtagctgtgcctgggcccttggaatcggctgaggccacggcaggtgccccaggggatgttcACAGCAACTGGAATGAGGTGAGGGCTTCAGCAGGGCCCCGAGAGGGACAGGCAGAATCTGGAAGTCAGTTCTCCACAGTCCTCTATGTCCCTTATCCTGCAGGCCCTGCTCCCTGGGTCCCGCCTGGATGTCTCTGTCCCTCGGAGTCGTGTGCTCCTCTC
This mutation is a 2414 bp deletion beginning at Chromosome 7 position 126,585,381 bp and ending after 126,587,794 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42625 | GTG CCT TCA CTC TTC TTT CTT C | Common | A | |||
42626 | CCT CAG TCA CCT CTC CCA GT | Wild type Reverse | A | |||
42627 | TGC AGG ATA AGG GAC ATA GAG G | Mutant Reverse | A | |||
42628 | Fluorophore-1 | AGA CAC AGG CAG CTG AGG A | Quencher-1 | WT Probe | ||
42629 | Fluorophore-2 | CTA GGA CTC ACA GCA ACT GGA A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42625 | 0.40 uM |
42626 | 0.40 uM |
42627 | 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 |