Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:37517492+37517638 147bp GCATGTGTGTGTGGCTACCT AGCATGACTTGGCAATTTGA
Mut= 147 bp
Wt= 145 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
ATTTTATTGATTTGATTGCTCCTTCAGTTGTAGACATTTTTACCGTGACAGAGTAAGTTAGGAAGGAACAGACTCTGTAAGTGACCTGTTTTGTTAGCTACTTCGTCTAGGCAGCTGTTTTTCCCCTGTACTTAACTAAGAGTAAGGAAGATGTTTTCCACTTGGGGAAATTCTTCTTTCTGTCCTCTTTCCCTCACATTTTCCAAATTCTGGGCACCATAGCAGCTCTCTCTCCTGTGGCAGGTGTACATCCTATTGGCTGGCTGGTATTTCTTTTTTTCCTCCCCCTTATCCTTTTTAAATGGGGGAGGGGGCATAAAATATgtgtattggatacatgcaataatgttgtaatgtttcttgttgttttatataattttataattgtaaattaattgtttgaattataacatgtttgagtaaatgctaaattagtatttttctaatatgataatgcatttgaaatagtattcctgtaacaatgtgtctgtgtttgtctgtctgtgtctgtctgatagtaattaatatctgtggtccgtatctggaggaggaagtacagctttaaaggaataacaaaagactttgtgtcttagacccttcgcaggtgttacagtcgggtgaaagaacatggtgttgggaaaagaaagagcagttacacatttgaacagttggaacaggtgtttggtcagggaggatgggatgctcagccctgccagcctgtacttattaacagtagtggcttgtaccaggagctggagtcagatggcagcactatggaggactattcacaggaggactggggaaaccacagtcaggagctccatggctatccaacagatcaggaattgggtaagaaaacaacatgtgtgcatgtgtgtgtggctacctgatagtgcaatgaatggtatttataccatctgtaatggcttctagtgttgtcagcacaaggcatttgccaaactgtttcagtcaacatggacatccttgatttaacatcaaattgccaagtcatgctcactattagctacgtggtgacttgcatactgttaagttccttgaagatagagacttcctaatctgagtcatacaggagGTTCACTGACACAATCTTATGCAATTCAGAATACCCCTGTCTGTTCTTTGTAGCACTAAGATTTCCTGCTGAGAGCACTGACTCTTGGCCCAACTTCTTTAAATCCACACTGGTCCAATTAAGAGGCTACATGTAGTTTTTGTTTGCTTGTATCCCTGTATGTCAAGTGCTCCAGTTTCTTATTTGGGAAGTATTGATCAGGTTTTAGCTATAGCCTGTGTACATCAGCAGCAAGTTGTGTAATGAGTTTTAGTAGGAAATGCCCTTCCGCCTAGTGAAGTGCTCAGGTGCCGCTGAGAAAGTTCAGGCAGGATTGGCAGTGTAAATTTGCTTCCCTTGCTTAAGGGAAAAAAATACAGAGA
This mutation is a 769 bp deletion beginning at Chromosome 9 position 37,516,948 bp and ending after 37,517,716 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49361 | ATC CTA TTG GCT GGC TGG T | Mutant Forward | A | |||
50035 | GCA TGT GTG TGT GGC TAC CT | Wild type Forward | A | |||
50036 | AGC ATG ACT TGG CAA TTT GA | Wild type Reverse | A | |||
50037 | AGC AGG AAA TCT TAG TGC TAC A | Mutant Reverse | A | |||
50038 | Fluorophore-1 | AAT GGC TTC TAG TGT TGT CAG CA | Quencher-1 | WT Probe | ||
50039 | Fluorophore-2 | ATG TTC ACT GAC ACA ATC TTA TGC AA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49361 | 0.40 uM |
50035 | 0.40 uM |
50036 | 0.40 uM |
50037 | 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 |