Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:46890543+46890692 150bp CAACGCGAAGAAACCAATAG CATCTTGACTCCAGGCTTCC
Mut= 98 bp
Wt= 150 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GCAGCTGCCGCTGCTGCAACTCACCCCACAATCCCGGATATAAGTCCTGCCCCTTTAAGAAGAAAAAAAAAAAAAAAAAAAAAGCTTCACCCGACTGCCTCCCACCCCACCCATCCACCAGGACCTTTGTCTCCCCACCCCCTCCTGTCAGCCGGCCCCGCCCTCCTTCCCGCCCCTCCAACGCGAAGAAACCAATAGGCGAGGAGaatatggagggagctgacgtcaccgcagctggccaatcacagcgcgcgttggtgggaggcctcgcgcggagagcgagcgctcggaggcaaggcggcgggggaggaagcctggagtcaagatggagggtgtggactgctccatggctctggccgacgctgccgctggctccccgcgggacctgagcctggtgcccgagcgacttcagaggcgcgaacaggagaggcagatagaggtggagagacgcaagcagaagcggcaggaccaggaggtggaggaggagaagagcggcttcttcgccgccgccttcgcccgggagcgcgcagcggtcgaggagctgctgcggggcgaggcgtcggcggagcggctggaagaggcggccaaccggctccagggcctgcggaagctcctgaacgactcggtgctcttcctcgccgcctacgacctgcggcagggccaggcggcgttggcgcagctgcaggctgtcctaacggaacgtcgccaggagttgcagcccaagaagcgcttcgccttcaaggcacggaagaaggatgctgcggggaccgcccaggtagacgccgcacccgtcgcctcggcggccccttctccaccggtgaccaaggaggaggagggagctcccggggccagctgggcctgtggcttctccaacctggagtcccaagacctggagaagagagctgaagagctgcaccagcgcgacgtccttttgagcgacctgaccaactgcactgtcaaactgtgtggcaatcccaacaccttgcggcttgcgaaggcccgcggctgcaaggtgctctgcggcccagtgaccacctctgtgtttctggaggattgtcgagattgcgtcctggctgtggcttgccagcagctccgtgtgcacactaccaaagacacgcgcgtcttcttgcaagtgaccagcagggctatcgtggaggactgcagcggcatccagtttgccccttacacctggagctacccggggatcgacaaggacttccaggactccggtttagataggagcaaaaataactgggaccaggttgatgatttcaactggctggcccgaaacgtggcctccccaaactggagtattcttcctgaagaggagagagacatccagtgggattgagcactggccacactcagtgtgatgtcccattcggtttacctgatattgtcttatgtttgcggtgataggggttagaaattgttgaaactcccctcctgtgagtcctattaaattcatagcagtgtaacagacttttgctggtttggccttcccctgcctccctttgagTATGACAGCGTGAAGAATTAGGGGGAGAATGGAGGAGCTTTGAGGGATTTGCTGTTCTTACCGTTGATCACAGGGAGTGATAAAGTGGGTCTTCAATAAATAATAAGGATGTCCGGAGAATTCCATTAGCGGACGGTTTAAATGGAAGAAGTAGGCCCATAAAAGCTCCATTAACGGCTGTTTTCCTCCCACTAAAGGTTTGTTCTGTGTCT
This mutation is a 1313 bp deletion beginning at Chromosome 17 position 46,890,571 bp and ending after 46,891,883 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50196 | CAT CTT GAC TCC AGG CTT CC | Wild type Reverse | A | |||
52533 | CAA CGC GAA GAA ACC AAT AG | Common | A | |||
52534 | TGA TCA ACG GTA AGA ACA GCA | Mutant Reverse | A | |||
52535 | Fluorophore-1 | AGC TGA CGT CAC CGC AGC T | Quencher-1 | WT Probe | ||
52536 | Fluorophore-2 | CGA GGA GTA TGA CAG CGT GA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50196 | 0.40 uM |
52533 | 0.40 uM |
52534 | 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 |