Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:136401853-136401985 133bp GCCTGGCTAAGAGACAACCTC ACTAGTGTGGAAGCGAAGTGG
Mut = 149 bp
Wt = 133 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
ATGAGAAAAATTAATTTCTTCTCATGGAGAAAATGAAGTCTCTAGCCTGGGTGGAGATCCCACATCAGTTGGGCTGGCCTATTGCCTGGCTAAGAGACAACCTCTTGGGAAGATGgattctccatgctaaaggggacagcaagaatatttattcctttctaagataatacatttttgatcaggcgtcttgccgggccacttcgcttccacactagtctcggaggagtggagaggaaggatgtcgtcaggtcttaaggctgagctggagttcctagcatccatcttccacaaggaccacgagcggctgcgcatcgtgagctggcacctggatgacctacagtgtcagttcctggtgccggaggcaactgtgggcagtccgccctcgccgccaccgctcacccttcactgcaccatcaccgagtcctacccatcttcaccggcgatgtggttcgtggactctgacgatccagatctcacatcgatcctggaacgcctggaaaattcgaagccagacagttcgcttagtcagcagctcaagtggctgatctgtgaactctgccgcttatataacctgcctgagcacctggatgtggagatgctggaccagccggtccccatcgctcagaaggccaccatcaaagatgggtcttcagaagaagacgaagatgcatcttacggactggacgactcggattattatgacatgaaagaagaagagacgagtagtgagaaaacgtcagagaatgaagggcttgagaaagaggatttggaaatattagagaatatccagaaaaacctttggcaaaagaatgcaagtggtgccacgtctggctcagtgtgtgcttcagatagacttatgaaagaactcagagaaatatacagatcgcagagttacaaatcagggactttttcagtggaactgataaatgacagcttatatgactggcacgttaaactgaggaaggttgaccctgatagttgcttgtacagagaccttcagcgcttgaaacaaaaagaaggcatagactatattttgcttaacttctctttcaaagataactttccgtttgatcctccatttgttcgagtcgtgttgcctgtactctctgatggttatgtcttggatgggggggcattgtgtatggaactgcttaccaatcagggctggagcagcgcctactccatagaatcggtcatcttgcagatcaatgccaccttagtcaaaggcaaggccagggtgcgctttggagtagataatcactatactgaacaagtggctagacgggtctataaatccatggtgctgaagcatgagaaatcaggctggttcactcctccacagaaagatgggtaaatagtagagcatcatgtatttggtatttgtagcagtagtgagaaagttattagctaattggtgctttagtggataatctctcaactgaccaccagtagaagtgtcagcctttccattttgcttactgtgggggtcttgggctgaactgtgagccttactgtggtttttcctgataaataatcataactccattttcagtcttccatgttccttgattaagtgaaatgtgttaagtaggccagtcagttgtgactttagataatgctggttattaggctactgggggggggataatgagtatttttaaacatttgttccatttagtactaataaaagtttttccagtgtccaatactgttttagaaaataaaataaaataaaataaaaaaagaataaaaaagaatacatttttttcttcccagaattcctgcccttgtgCAGTTGCTATGGTGTCAACTGTTAGGCCTGTCCATCTTTTTAAATCCTGACTCAGACAGGTACAACTTTATCTTTAAAACACAAAACAAAAAAATAACAAAACCCAACCAACTCCTTTTTTCTTTCTTTCTTTTTTTTTTAATCTGACAGACAGCAACTTTAT
This mutation is a 1677 bp deletion beginning at Chromosome 6 position 136,400,277 bp and ending after 136,401,953 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50611 | GCC TGG CTA AGA GAC AAC CTC | Common | A | |||
50612 | ACT AGT GTG GAA GCG AAG TGG | Wild type Reverse | A | |||
50613 | AAG GAG TTG GTT GGG TTT TG | Mutant Reverse | A | |||
50614 | Fluorophore-1 | CAT GCT AAA GGG GAC AGC AAG | Quencher-1 | WT Probe | ||
50615 | Fluorophore-2 | AGA TGC AGT TGC TAT GGT GTC AAC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50611 | 0.40 uM |
50612 | 0.40 uM |
50613 | 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 |