Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:34401957+34402046 90bp TCCATGGCCCAGCAAAGT GTGGTGCGGTTGTTTCACA
Mut = 99 bp
Wt = 90 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CCAGGACAAGCGTGCGGTTCTTCTCAGTGAGGCTCCATGGCCCAGCAAAGTATgaaagtgaggcctgtgctcctaaagcgaagcagccttgagtctgtggagcttgtgaaacaaccgcaccaccgcaggagcaaatcgcagcaagtgcgattcaaggaagatggcaacattaagaatccaactggcgtgaccgaggtcaacacccaaacccccgaagacccaggtgtaatggggaaggcacaggcctccaggcaccatcatcctaccacctactcgctgtctttccccaggtcccacaaggcaggaggattccgcagcatctccatccagacctcccccagtctcaggaagcatttccccgttttcaaaaggaagaaactcacgactagcaagtccctggtggaaatgccaacagcatccccaagtgccatccaggtcaacggcaacctatctgaacaggatattgtgtcctctgaccttgccttcctgaggctggctcagcatcttgaggatgggcctcgcaggctcaaaatcccccacccatttctccctaggatgcccaaggtgcaaagcaatgggcctgtcagcttctgcttagaatctgggacttggatgtcctcagagaaagccacagctgctattcaggttccagatgatatttgtcacagtccaacctgggaagctagagagtcggctctcagcccagaaagctcagctgaagagagcaactctatacctgctttgatctccatgtgtccaggggatgggcaaagagtgatgacgtcagagctggaaagaatgcccccctgctcaaataccaatagcagtgccagtaatatgccgggcacagaaaaactcacacccgaattgcttttgcccaaagacaaccctgatgacaaagaccttggccttccatcatctcagtcaaagaagatgtgtgttccctcaccaccacggactcacagttcgcctgagccaggctcccgtagccagccagtccacctaggaagatcttctgattgcccagcctcaggtgacaatcaccaggacctggagtcactcagaagtagcagtgcatccaagtctgtccctgtgtgctgggaacatgtgacaaagctccccagccagtcagacaccccagagcttcagactggtgttggaagtgagcaactccctgcctcgattccaaggcaggagaacagagctcagagcagcagggagataggtgggagtaatcacagtcacctggcgcagggagagctctgtgacctccaaggccggctgcaatcagtagaggaatcgctgcactcaaaccaagagaaaattaaagtcctattgaatgtgattcaggatctggagaaagctcacgctctcactgaaGGGTAAGGTGAATTTCTTTATGTTGAAAAACCACTGAGCCGAGTTGTTTTCTGGGAAGGGAAAGAATGAGAAAGCAAGGTCTCCCACAAAAAGTTCTTGCTCTGTTGTTTACCTAGACGCTA
This mutation is a 1327 bp deletion beginning at Chromosome 11 position 34,401,977 bp and ending after 34,403,303 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51552 | TCC ATG GCC CAG CAA AGT | Common | A | |||
51553 | GTG GTG CGG TTG TTT CAC A | Wild type Reverse | A | |||
51554 | CCT TGC TTT CTC ATT CTT TCC | Mutant Reverse | A | |||
51555 | Fluorophore-1 | CCT AAA GCG AAG CAG CCT TG | Quencher-1 | WT Probe | ||
51556 | Fluorophore-2 | ACT GAG CCG AGT TGT TTT CTG G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51552 | 0.40 uM |
51553 | 0.40 uM |
51554 | 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 |