Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:123845651-123845767 117bp AGCTCCTGCTACGGAAACG CCAGCTATGGAAACAAATTACCAG
Mut= 123 bp
Wt= 117 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GAGTGACTTAGAAGCCAGTCTTCAGCAGCTGCCCAGTAAGGACAGCCTAGTCTGTCCGCAAAGGAAGCATCCAATAGAGAAGCCCTCAGGGGCCTGTGGAGGGAGCTCCTGCTACGGAAACGCTGCTCATTTGGATAGCGATGGTGActcctgtggatgggcatcacgggtgctttggaatagttttggtttaatgctggtaatttgtttccatagctggtttaagtaaaacctgtagagttaaacatgtgactgacttgatgaaacattatttttcaacttaagtcatatatatgtatatatgtatgtgtgtgtgtgtatatatatatatatatatatatatatgtatgtatgtgtatatatatatatgaacttgaactttaaagtttttccatctcatcttgtcccttttttttttccaaaagcaccaccaaaaaaggacacggaagagaaacccgaagaacacaagacaaaagagactgatggggcacccagtggccctggaccgcggcaacagcctagtgccttatgtgccagaggaagtaagaaagccaccagaagcccacagagatcgacgagcaagataaaagagaacaagcacccgtttgctctttatggctggggagagagacagatggacatgggaagccagaagacacacaacgtctgcgcgtcagcctctgtgcacgaggtgtgccttgaacggggccagactagccctgagctgccttgggggcatttctcctatggctacggtgcccagcgccactggaagtccttgggttatgtgggagtccatggaagccaaaagaaggggtctctggtcctagaaagatggagtggactttggttaaCTGGGGCTACAGCCAGGGCTAAGGTCTGATGACAGGAAGAGGTAGCTGTCATTGTGGTGTATGAGTTCTGTGGTCTGGGCCACAGGGATAATGGGAGCAGGAGTTATCCCCTGGGGAGGCATC
This mutation is a 707 bp deletion beginning at Chromosome 8 position 123,845,017 bp and ending after 123,845,723 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43583 | AGC TCC TGC TAC GGA AAC G | Common | A | |||
43584 | CCA GCT ATG GAA ACA AAT TAC CAG | Wild type Reverse | A | |||
43585 | CCC AGA CCA CAG AAC TCA TAC A | Mutant Reverse | A | |||
43586 | Fluorophore-1 | CCT GTG GAT GGG CAT CAC | Quencher-1 | WT Probe | ||
43587 | Fluorophore-2 | CTG GGG CTA CAG CCA GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43583 | 0.40 uM |
43584 | 0.40 uM |
43585 | 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 |