Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:24850316-24850451 136bp CACGCCCTCAATCCATCAG GCTAGGGCAGCTAGGGAACA
Mut= 106 bp
Wt= 136bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
ACTTACCCAGACCACGGCGCGCGCAGGTGGCTCCCAGCGCTGACAGGCTCCGGAGACACAGCGACCCGCGACCCAGCACCATGACCCACGGGGTGCACAGCCGGGTCGCGGAGTCCGCAACACCAGCGTCGTGGGCATCAACACTGGCGTTGGCGACTGCAACCAATCAGTGTCGTCCTCCTACTGCTCCCAACCAACCAACTCCTGCTCTTTACCAAAATGCCACGCCCTCAATCCATCAGTGACCGCCcatagagttcacagccaaccagtgcctgtctcgcaccgccgcgccgcccctagccaatcagtagtagctcctgccccccccccccaaaatgttccctagctgccctagcagccttggcctgtagactcggtctgcaagtgggtgttggaggaggctgagcgtgagcctcctcgtctctctggggcagtcgcctgcacgcagagacctttcgctgacctcagcgtcccgctgctgcgcaaggaagggcggggccgctccgacctggacagcgtccgaagccagtcctctggaggccgccgtagtccgggggagtcggtggtcacatgacccaaagctgtactatggcttccaccaaaccgctgtctcgtttctgggagtggggcaagaatatcgtctgcgtggggaggaactatgcagatcacgtcaaagagatgcgcagcaccgtgctgagtgagcctgtgcttttcctgaagccgtccaccgcgtacgctcccgagggctcaccggtgttaatgcccgcttactgccgcaacctccaccacgaggtggagttgggagtgcttctgggcaagcgtggtgaagcgatcccggaggctgcagccatggactacgtggccggctatgccctgtgcctggacatgactgccagagatgtgcaggaggagtgcaagaagaaggggctgccgtggaccctggctaagagctttacgtcctcctgcccggtcagtgccttcgtgcccaaggagaagattcctgaccctcatgccctaagactgtggctcaaggtcaacggagagctcaggcaggagggcaaaacatcatctatgatcttttccatcccctacatcatcagctatgtttctaagataataaccttggaagaaggagatcttatcttgaccgggactccaaagggagttgggccaattaaagaaaacgatgagatcgaggccggcatagatggggtggttagtatgaggttcaaggtgaaaagatcagaatactgagagtagagtgccaaaggggaagggagacagaagcaagggaaataaatgacactaataatgaaaatctaaaaattatgctagacatgtcaaaaaagatgaatccttaaaaaataacgtgatctaaaagacctgggactgaaatagaaacaggaacaacgaagctaaaggatatagaatgcttcccaagagggcaatgttaaactaaactttgagaagttgtagttttcttttctaaaactgaactgaatagacttttcaataaatcattctgaagtctatacctccagacttaaaagtgcaaaggaagtaactcattaatgctgttggcctttaggaaagctgtttgtacagagcactccagttgatttttacttgggaaagtcctccacctcaaagccagataagaattttgttaagtgagctcttgctgcagccctgccttgtttactttgggtgtggcttctgagtttggaagataattttgaaattgttttccaaataaagaccatttctgtgtgactgaaaagagcactttcttttcccagggtggggagcacttcttcctctgggacttaggaccttggcctttggtttcctgagcactgtgcctctttaacttattaaccacataaaccaatacttccacctcaaagacacaggccctagggaactgacaagcctggagatgctgtgtgtgttcatgcttgcacccctccctgataaaatgcctgtcaggatgcacagcacagttgtcagggcctagtggagtcttgtagaattagatagtggcttttgctgagagggtacataggagcctgctggtccctttaagaaggcatgttacatcttgttagtagtttatcattactgtaaggcaatatgccaaccatagttcattccatatttctgtgtattctgggtttctTGTTTGTTTTTTTTGTTTGTTTTGCAAAGCTATACCACTGGCATTATTATTATTTCTGATGACAATGCAGACTTAGAAGATAGAACTGAAGTAGAGCCAAGTTGTAATGAAATTGTCACAAACTCAGAACCTGAAGTTTTAACTACAGCATAGGTAGTGTCATATAAGTAGATATATAAGAAAATTTAAGCATTTAGGTATTCTGTGAGCCCCATGCTTAATTATTTATTATGAGGTCCCATAGTGCTAT
This mutation is a 1932 bp deletion beginning at Chromosome 17 position 24,848,493 bp and ending after 24,850,424 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50866 | GCT AGG GCA GCT AGG GAA CA | Wild type Reverse | A | |||
52524 | CAC GCC CTC AAT CCA TCA G | Common | A | |||
52525 | CTT CTA AGT CTG CAT TGT CAT CAG | Mutant Reverse | A | |||
52526 | Fluorophore-1 | ACC AGT GCC TGT CTC GCA C | Quencher-1 | WT Probe | ||
52527 | Fluorophore-2 | TGC AAA GCT ATA CCA CTG GCA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50866 | 0.40 uM |
52524 | 0.40 uM |
52525 | 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 |