Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:34897088+34897222 135bp CTGGGGAAGTGGGAAAGTAGA CCCTCCGCTACTCACTGTTC
Mut = 105 bp
Wt = 135 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CCACCCCTTTATCCATTCTCCCGTGGGCGCGCGGGCTGCGGGACTGGGGGGTGTGGACGCCGCTGCCCCGCCCCCGGGAACCACGCCTCCCTCCTCGGAGGGACCGCGCCTCCTTggaattggggaaactgagggaggcgcgccaagactagggaggtagggatgaggggtctggagccttggcctccgacagccagagacctttgtctctgactcgtttccttcccacccagggcagtccactcccgccacaatggcctctggggtggaagtcctgcgcttccagctgcctggccacgaggccgcaacgttgcggaacatgaaccagctccgtgcagaggagcggttctgtgatgtgaccatcgtggccgacagcctcaaattccgtggccacaaggtcatcctggccgcctgctcaccgttcctccgggaccagttcttgctgaaccccagttctgagctgcaggtctcactgatgcacagcgcgcgtattgtggcggacttgctcctctcttgctacacaggcgccctggagttcgcagtcagggacatcgtgaactacctgacagcagcctcctacctgcaaatggagcatgtagtggagaaatgccggaacgcccttagccagttcattgagcccaaaataggccttaaagaagatggggtcagcgaggctagcctcctaagcagtgtcagtgccaccaagtccctcctccctccagccaggaccccaaagccagctccaaaacccccaccaccacctcctctaccccctccgctcctgcgaccggtgaagctggagtttccgctggatgaggacctagagctgaaggcggaagaggaggatgaggatgaagatgaagatgtttctgacatctgcatcgtcaaagtggagtcggctctagaagtggcacaccggctcaaaccccctggaagcctagccgggggtctgggtatcggggcctctgtgagcagccacctgggagagctggcccagagtagtgtggcccccaacactgttaccccaccgcaaggggtggtgaaggcatgctacagcctgtcagaggacgcggaaggggaaggcctgttgttgatcccaggaggccgggccagtgtgggggccacctcgggcctagtggaagcagcagcggtggccatggctgcccggggggcggggggcagtctgggggcagggggtagccggggacctctgcctgggggcttctcaagtggaaaccccttaaagaacatcaaatgcaccaaatgccctgaagtgttccagggtgtggagaagctggtcttccacatgcgtgcgcaacacttcatcttcatgtgcccacgctgcggcaagcagttcaaccacagcagcaacctcaaccgccacatgaacgtccaccgtggtgtcaagtcccattcatgtggcatctgcggcaagtgctttactcagaagtccacactgcacgatcacctcaacctgcactcaggagcccggccctaccggtgctcctactgtgacgtgcgctttgcccacaagcccgccattaggcggcacctcaaggagcagcatggcaaaaccacagctgagaacgtgctggaagctggtgtggctgagatcaatgtcctcatccgctgaaaggccaagaggctgggggcggtcctgggcccggcagaattggggaggggcggggtttgagggcaggcaaacattttgagggaggaagactggagagagagagagaccatgcctcaaagagcagaagaaaaagatggagccggagacagggttgataaggctgagggaatgtgggatcccaaagaaagatttcttttttgaagaggtgcataaacgatgtggaggaagagagggaaaaaaaaaaaaaggcaaagttgaaagttttggagaacagctctttcctggctaaagctggctgggggggggggggattctagcgtttcttgagatggggtgggggtgggaattgtagggggattgccaggaagcagcttacttgcccctggttagaatagatgacagggagaagggagcggagggcgacacatgtatgaagtaccagaaggaagggaggagatttttgcatgctggggaagtgggaaagtagatacaaggctcaccggaaatgaaatgatatgtgccaggtaagagaacaCGGTGGGCAACCTGGGTAGGTAATGAGACATTAGGCTACAGTAGCGAGAACAGTGAGTAGCGGAGGGGTGGGGGGGCAGGGCACTTAGAAAAAAAGGACACAAGGAGGGCTGTTGGGGGGAGGGAGAGGAGTAGAGACATAC
This mutation is a 2053 bp deletion beginning at Chromosome 17 position 34,895,103 bp and ending after 34,897,155 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51306 | CTG GGG AAG TGG GAA AGT AGA | Wild type Forward | A | |||
51307 | Fluorophore-1 | CAA GGC TCA CCG GAA ATG AAA T | Quencher-1 | WT Probe | ||
51759 | CCC TCC GCT ACT CAC TGT TC | Common | A | |||
51760 | GAA CCA CGC CTC CCT CCT | Mutant Forward | A | |||
51929 | Fluorophore-2 | AGG GAC CGC GCC TCC TT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51306 | 0.40 uM |
51759 | 0.40 uM |
51760 | 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 |