Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:112504549-112504656 108bp CTTCCTGAACCTCCTGGTCA CCCTTCTCAGTGATGGTGTCA
Mut = 102 bp
Wt = 108 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TGAGTTCTTGACTTGGCCCTGGTTCCCCGGTACATCAGTGGTGGCAAGTCCCCAACTCCCAGTCTGCGAGAGAACAAGATGGCAGCTCAACATCGGAACatttgcagggatggcagcagaggagatggaagcagccctctgagccccattccatcccatcagggccccaggtgctcaccagaaaacctggacatcccccctcctgtttcaaggggagctatttggggtcagaccttgagtggctccttcagcatccatctcttgactccactgcagctccttcttctacgccttcctgaacctcctggtcagtgcctttgtggtcttcctggtcttcattgccagcaccatcgtgagtgtgggcttcaccatgtggtgtgacaccatcactgagaagggctccacaccccacaggtatgcctcacccctgcttcatcagccacctacaagacccacctttacctgggtccctatgtttttcttcctgaatctgaggctttgcccacctgtgccccacccctacacctgttgccttttccctaagacccacccacctgcgccccacccccgcacctgttgccatgcccctaagacccgcccacctgcgccccccccacacctgttgccatgcccctaagacccgcccactcaatgtcaggcacctctttggtctagtagccctacagcattacacatggaaggtccctgtgggccgagaggcaccagatgggatcctgcagttgacaccgcctggcacacttgctagccttagctcgctctgaagctaactccactgtgtcgccatgatgttagtttgtggttggtgagtggagattgccctcatggtgtccctgtgacacttatgcagggacacagtgcaatgtgcctcagggggcatcacatccatctcagactgggaactgcaggactgactagacagagcagctcatgaccccgagtgccggacagacagtgacaagatagtgctccgggccagtcgtaaaggtctgcagctcctcttctttgaagcctgtcttgctccacccagtgaggggccaaagctgaccccgagtggagttcctagaggcatctgtccctttgggtactggcccctctgtcttagggttttgactgctgcaatggaacaccccggccaacgcgtcttggggaggaaagggtttattcagcttacacttcaatatcacagttcctcatcaaagaaagtcaagacatgaacacagcagggcagggacctggaggcaggacctgaggcagaagccactggaggggtgctgcttactggcttgctcctctagatctgctcagccaggggtggccacgcccacaatgggtggggctatttcccaccaatcactaagtaagaaaatgtcttacaggcttgtctgcagcctggaagcattttctcaattgaggttcctgcctctcgactttagcttgtatcaggttgatctaagacgccactcagtggttaggggaagtattctctgcatcctagctctgctgccttttgacttgggggctcaggggctcggggcctggggggctcgggggcttgggggctcaggggctcacaagtcaccagccacttggcttcgtgctattttagctgcgaggagttccaggaaacggacttggagctgaacgtcgacaactctgccttctaccatcagtttgctatcgcccaggtaggcggggtctcgggcaggtaggcggggtctgtctctggccggaagggaatggctctgttggggtgaggggtgagacctcgacagatagaacagactcccggatcccagataaggaaacagactcagatacggtggggccatgCCGGGGAGGTGCAGGCTGTGGAGCAAGAGATTTCCTAGGAGAGGTTAGGAGAGTGGGGTCTCTGAGCAAGACTTGTGTCAGGGGTTGGGAGATAGGGGAGTCAGCAAAGGCAGAC
This mutation is a 1752 bp deletion beginning at Chromosome 12 position 112,503,097 bp and ending after 112,504,848 bp (GRCm38/mm10). There is a 3 bp insertion (CTA) at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48467 | AGA GAA CAA GAT GGC AGC TCA | Mutant Forward | A | |||
48470 | TGC TCA GAG ACC CCA CTC TC | Mutant Reverse | A | |||
51461 | CTT CCT GAA CCT CCT GGT CA | Wild type Forward | A | |||
51462 | CCC TTC TCA GTG ATG GTG TCA | Wild type Reverse | A | |||
51463 | Fluorophore-1 | CCT GGT CTT CAT TGC CAG C | Quencher-1 | WT Probe | ||
51464 | Fluorophore-2 | AAC CTA CCG GGG AGG TGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48467 | 0.40 uM |
48470 | 0.40 uM |
51461 | 0.40 uM |
51462 | 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 |