Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:62119483+62119590 108bp AAACCCACCCAGATTTCTGTA ACACAGGTCACCCCTGACG
Mut= 142 bp
Wt= 108 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AGAAACCCTATTCAAAACAGATTTTGAGCATGTCACTGCAGACACCCCATCTCAGCCCCTGTCAGCCGCTAGCAACCCTTTTAAGAGCTCCAAGGGCTCCCCTACTGGGAACTCCCCAAACAATTCAAATGAGTTGTCCTTGCCGCTCTTACTGAGAAGatacctagaggaggaagaattggatgaagagtgacagccatagcatcatctatgccatatatggaagacatttgagtttcattataaaattttttttctttcttttttgtttcattttttctttcatcttttgtctttagggactattccaacaactaaacggacaggcattccagctccacgggagctctcagtaactatctccagagagaggtctgtgccacgtggtccctccagctcaaagaagttaggctccagcccaacttcttcctgcaatcccactcctaccaagcatctgaggaccacccctgcaaaacctaagcaagagcatgaaggtgcagagaaggctgtgctcgagtcccaggtacgagaacttctggcagaagccaaaaccaaagacagtgaaatcaacaggcttcgaagtgaactaaaaaaatgcaaagagaggtgggccctgagcactgaggatgccaatgcttcagacccaagtgcagagggaacagcatccccagaaagtgatgcacagcctttgataagaaccctggaggagaagaacaagacctttcagaaagagcttgctgacctagaggaagaaaacagggccttgaaggagaaactgacttaccttgagcagtcaccaaattcagaaggggcagcaagtcacactggtgacagcagctgcccaacatctataacccacgagtccagttttggaagcccagttggaaatgagttgtctagtgaaacagatgagtacagaagaaccacacatgggagtgccttgcggacatcagggtcttcaagcagtgatgtcaccaaagcctctctgtcaccggacgcctcagattttgagcatatcactgcagacaccccgtctcggcccctgtcagccactagcaacccttttaagagctccaagggctcccctactgggagctccccaaacaatgcaagtgagctgtccttggcatctctcacggagaagatacagaagatggaggagaatcagcacagcactgcagaagagctgcaggctactctgcaggaattatcagaccagcagcagatggtgcaggaactgacagccgagaacgagaagctagtggatgagaagaccattctggagacctccttccaccagcaccgagagagggcagaacagctgagtcaggaaaatgagaagctgatcaaccttttacaggagcgagtgaagaatgaggaacccagtgcccagggagggaaggtccttgagctggagcagaagtgcacggatattctcgagaagagccgctttgaaagagagaagctgctcaacattcagcagcagctgacctgtagcctacggaaggttgaggaagaaaaccaaggcgctatagacatgattaagcacctgaaggaagaaaatgagaagctgaatgggttcctagaacatgagcggtgtaataatagtgtgatggccaaaactctggaggaatgtcgagttaccttggaaggcttgaaaatggagaatgggtccttgaaggctcttttggaggctgataagcaaaaagccatagaggccagcagtactgtggggcagacagcagagaacttcgaggttcaggagatgctgaaagtggctcgagctgagaaagatcagttgcagctgtcttgcacagagctcagacaagaattgctaaaggcaaatggtgaaattaaacatgtttccagcctgctagctaaggtaagaagacagttctcaactagtatttgttgacatttctctttgagaccttaacattgtgaaccaaagagaatcaattattcaactgtatttgtgaatctaagccttctgatttggaaatctattagaatttaggcaggttagctctggggattccaaattttaaaacaaaacaaaacacaaaacaacaacaacaaaaaaaacccacccagatttctgtacagatagataaaaagattagtggaaatactgtaaattacatcagatgatatggtgtagTGATGAAGACCGTCAGGGGTGACCTGTGTCTTGGTGGTAGAATACTTTCATAGCCTCCTTGAGACTTTAGATTTGATCCCTAGCTCCACACAAAAATAGGATAAAATAAGATTTAGAACCATCCTGTATACATTGATGGACTTTTTGATACAT
This mutation is a 2002 bp deletion beginning at Chromosome 11 position 62,117,560 bp and ending after 62,119,561 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47023 | AAA CCC ACC CAG ATT TCT GTA | Wild type Forward | A | |||
47024 | ACA CAG GTC ACC CCT GAC G | Common | A | |||
47027 | CCA TCT CAG CCC CTG TCA | Mutant Forward | A | |||
47028 | Fluorophore-1 | AGT GGA AAT ACT GTA AAT TAC ATC AGA TG | Quencher-1 | WT Probe | ||
47029 | Fluorophore-2 | AGA GCT CCA AGG GCT CCC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47023 | 0.40 uM |
47024 | 0.40 uM |
47027 | 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 |