Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:11579360+11579461 102bp GCATACATGCGTGGAAATGT GCTCCAGATAGGATTTAATGCAC
Mut = 91 bp
Wt = 102 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TTGTAAACAAATAACACAGAACAGTTCCTCTTTTGTTTTTATTAAAGTGACTGCGGTGCTGTTTCAGCCCCTCTGCCAGCATACATGCGTGGAAATGTGATATTCTCACTAAATTAGCTcattttaagctgtggttcctgcttatctccagacctttgtgcattaaatcctatctggagctgctccctgtatacacaagtgtgttttgcttgtgctctcctgccttctgtgagaagctagctttatgtaggtcaggcttggcttaagcggctttcctgaacctgcgtaagtcagcttaagctggaggaattttaataagccctcccctgtaggcgtgggcctaaatgaggctagcctagttaagcctgatctttctgtttgtgaaaagagctgagcagagctgaaggctgcatggtggtttcagaaacggcctacttaattccaaaagaacaatggtaggaaaggctagatcttccaattttaccttatctgaaaagcttgatttgctaaaccttgtgaagccatatgtgaaaattctcgaagagcacactaataaaaattcagtaatagtggaaaagaatagatgttgggatgtcatagcagttaactataatgcgattggagtagaccgccctcctcgcacagctcagggcctacgcagcctttacaaaaggctcaaagaatatgccaaacaggagctattgcagcaaaaggaggcccagtctgagtttaaaagcagtgtttcggagcccaccaagcaagtcatggaaatgattccccagattgccagcttcggcctcataagagacaggagccacatacagaggtaagccttattttattttgttgtgggaattatggcgcctcctatctcttgggctctggcttagctgtgctagacatgtgcatagcgagcgtccccacaaggggagccgtgaacagtgtctttgttttgctttcttaaaaaaaaaaaaaaaaagtaaagctctgaggagttctctgttaggacagcttgcagctgctttccctccatCCTGCCGATTCCACAAGTAAAAGTTAAGGCAGAATTGATGGGATGAAGCTGTGACATCACTAAGTGAACTTTAAAGCCTTCATAGCGTAGAGTGTATAAAGTAATGGGTTTTACTATGACATTTTTA
This mutation is a 914 bp deletion beginning at Chromosome 4 position 11,579,401 bp and ending after 11,580,314 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50616 | GCA TAC ATG CGT GGA AAT GT | Common | A | |||
50617 | GCT CCA GAT AGG ATT TAA TGC AC | Wild type Reverse | A | |||
50618 | AGC TTC ATC CCA TCA ATT CTG | Mutant Reverse | A | |||
50619 | Fluorophore-1 | AGC TGT GGT TCC TGC TTA TCT CC | Quencher-1 | WT Probe | ||
50620 | Fluorophore-2 | TTA GCT CCT GCC GAT TCC AC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50616 | 0.40 uM |
50617 | 0.40 uM |
50618 | 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 |