Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr14:101199487-101199588 102bp GAGGGTCTTGAGGTCCCACT TGCGGAGGAAAAGATTTTGA
Mut= 94 bp
Wt= 102 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
TGGGATTGCAGGCTCATGCCAGCATGTCATGCTTATGTGGTGTTGGGTATCATACCAAGGACTTCACGAATGCTTGGCAAACAATCTACCTGCTGAACAACAGCCAAAGACCTCACATGGAGGGTCTTGAGGTCCCACTAGAAATGAGCACCTGAAGCAGAAtccaggagtgggccaatcatccctacattttgacctctgtcaaaatcttttcctccgcaggtcctacaggctgtgtcttcctcctgattccatccagatagttgtggttgtcccttagctcctcaggaacagccctcatcctctgtttccgtacgcactgcacctccagcgaatggctaaggagacgtgaggagccaagcttagatgcaggctctggactgagcacgccacagccaggtagaagaggctgcgtgtcctcttggcctgctgctgatgcctgcagctgttgaggccatactgtgcacagaaggcagtcttgaccaccagccacagctggcctcagagatctcccacgctgactgttgggtccctaaccactggtcttgtccaccccatccacttccctcctggattcaaggcaatgttgcctgtaaacaaggaccaggtgctgctacagaacacagtacctccagggtgtccccctcaggtcctctcacagtttgtgaattcccctgcccccaacttagcatctctttgtccccatccgaccctcccttcttcccactttcctctgcctgcccccccacaggcttatttcttctcttctttgactcagactcactctcctgggcctcacttctcttcggactcaaattctgactttgttcctcctcactcttctagtcacccacgatcctcttcttgctttggccaaaactatacttattttggtgaaaaactcccctctccacactccatctctccctctaactatcagctctgtgtgtctccccctctcaccggctcctcctctctttcccagctacaacactcctctcctcactcctgtcagtccccttcccgccttcaggacttacaaagccccaagatcacttccccggtccccagctcaccttctccaaggattcagaataacaagcaaacatggcagtggcctcagtccggaagcatcaagtcttctcggggggcaggggtatgtgtgccaagcaaggtggaccctgcagaattcaaggactcagggaccctcacccaagccctggtggaccatgtggggcgccgcagaattgcacgagacctgcagatacagtttctgcagcgcctgtggctcggcacacccggccatgccccagtcgtggagtaccccatatgtctagtgtgcctccagatccgcaccccatcttgtcccacccccaagtacaagactgtaccccagctgcttgccttccctcaactactaccctgtgtccagggccaggaatctgggcctctccgaataggaattggcttcggcctccgcctgcctcggggccaggccagggctttgcatctgctgcccccaaaaaactctactccggtgggagtagagtctcaggaagaggccctccagcgacaaaaatccacaatccaagaatcagttcaaatcaccggcacactcttccaggcccggagcctccggtctatagaccttcaatcaccaaaacccagccagtgttccaggtctctacttcaggaaccaagacaggtcgctgcatccccaaaggccgggccttctgtctcaaagaggtctgtgactctagggtctattctccgaaagtcgccatcttaatcccagtgcagcgaagaccctctggaacaccacctccaaactctaccttccctaccaggacttctcagaccttggagggctggctggctggaagccaggtgtgttctcctgccctgaaccctgggaaccccttgtggagtttgctctcccctagttgaagagaccagaggcacccaaggcctcatttagccttcagtgtatccaataaagcctaactccgagaatcttatgtttgtgtctactatgcccacaggatggaatcaaggctatgggataaagttagtccgacaaaagacttggcgggggggcgggggggcgcggagaaagattaaggataccaactGCTATCACTTGAGTACATGCTAGTACCTGATGCTTAGAGACCTCCACAAGGCCCTTCTCCTGTTTAACTCAAGTCTGTGGACAGATAGTTGGAATCAAAGATTAGGCCAATGGGTCTATTTCTCCAGGGACAGCCTGGGTTGACCAGGATGCAGCAGCTCATTTTCCTCACAGGGAAGGACATTGT
This mutation is a 1970 bp deletion beginning at Chromosome 14 position 101,435,012 bp and ending after 101,436,981 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
64695 | GAG GGT CTT GAG GTC CCA CT | Common | A | |||
64696 | TGC GGA GGA AAA GAT TTT GA | Wild type Forward | A | |||
64697 | CCT TGT GGA GGT CTC TAA GCA | Mutant Reverse | A | |||
64698 | Fluorophore-1 | CAA TCA TCC CTA CAT TTT GAC CTC | Quencher-1 | WT Probe | ||
64699 | Fluorophore-2 | CCT GAA GCA GAA GCT ATC ACT TGA G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
64695 | 0.40 uM |
64696 | 0.40 uM |
64697 | 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 |