Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:65390272-65390429 158bp TGTATCCACGTTGGTCTATGC AAGCTCACAACTCTGGCAGT
Mut = 164 bp
Wt = 158 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GGTATTTATAACCCCCAGGGAAGTTTTTTTTTCCCCTCCCCGCCCCGGGGTAGCCCCAGAGTCAGTGGCCCCTGGCAAATCTAGGCCCCCCAGCGGCAGCAGCCTCCACCCCTCTGGCTCATCATGCCCCCAGGCCccgaggtcccagtgcaggtgtgggtggacggtcaactcttccaggcggagcagtcgctgctggtggagcactgcggcttcttccgcgggcttttccgctctggcatgcgggaggcgcgcgccgcggaggtgcgcctgggcgcgctgagcgcctccggcttccgcacggcgctgcgggtgctgcgcggagagcggccggcgctggcggccgaggacgagttgctgcaggcggtggagtgcgccgcgttcctgcaggcgcccgcgctcgcgcgcttcctggagcacagcgtcacgtcggacaactgctcgcttttgtgcgacgcggccgccgccttcggcctgcgcgacgtgctccacagcgctgcgctcttcatccgcgacgggtcgcacgagctggtggcacaactggagctgcccgaggcccgagcctacgtggcggcgctgcgacccagcacctacgtggcggtgagcacgcacacgcccacaccgggcttcctagaggatgcgtcgcgcaccatgtgcttcctggacgaggaagaggacgcatggcgcacgctggccgcgctgcccctggaggccagcacactgctggcgggtgtggccaccctgggcaacaagctctacatcgtgggtggcgtgtgtggtgccagcaaggaagtagtggagctaggcttctgttacgacccagagggtggcacctggtgcgaattccccagccctcaccaaccgcgctatgatatggcgctggctggcttcgaaggccgcctctatgccattggtggcgaattccagaggacacccatgagctccgtggagtgctatgatcccgccacaggttgctggagcttcgtggcggacttgccacagccggctacaggggttccctgtgcccaggcacgtggccgcctcttcgtgtgcctgtggaggccggcagacatcactgccgttgtcgagtatgtggtgcaaatggacaagtggctgccagtggctgaactgtgtcgttcacagagctacggtcactttatggtggcccatcgcgatagtctctatgtagtacgaaatggaccttccgatgatttcctgcactgtgccatcgattgcctcaacctggtcacaggccagtggacatcgctacctggccaatttgtcaacagcaagggagcactcttcacgtctgtagtgcgtggggacaccgtttatactgtcaaccgtgtatccacgttggtctatgccattgaagatggcacctggcggctgctcagggaaaaggctggctttcctcggcctggctctttacagacctttcttctgcggctgcctcctggcactactggacctgtgGCCACCGCACTGCCAGAGTTGTGAGCTTGGAATGACTTCAGAAGATAACACAAGTCTCCATGAACCTTGGCGGTGTCCTCATGAGCCTTGACTGTGTGATTGGGGAGCTCTTGGGAACTCCTTTTTCCCTATTA
This mutation is a 1340 bp deletion beginning at Chromosome 9 position 65,390,300 bp and ending after 65,391,639 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50922 | AAG CTC ACA ACT CTG GCA GT | Common | A | |||
50923 | TGT ATC CAC GTT GGT CTA TGC | Wild type Forward | A | |||
50924 | GGT ATT TAT AAC CCC CAG GGA AG | Mutant Forward | A | |||
50925 | Fluorophore-1 | CTC AGG GAA AAG GCT GGC TTT | Quencher-1 | WT Probe | ||
51949 | Fluorophore-2 | CCC CTG GCA AAT CTA GGC C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50922 | 0.40 uM |
50923 | 0.40 uM |
50924 | 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 |