Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:103228230-103228319 90bp TTCAGCTACCCCAGCATCTC TCCTGGGAACAGATGAATCC
Mutant= 106 bp
Wild Type = 90 bp
Wt Sequence: TTTTGATGTGGACCTTCAGCCAAGGCAAAAGAAGAGCAGGTTGTCACCTTCAGCTACCCCAGCATCTCTCTAAGCAGCccagggttaggtgggaacatggcagaccgttggggcgaacggattcatctgttcccaggatcggagtgcagcagagccctgtgctctgtcatgactgccaccagggatgagaggagggtggaacagaggagaggtggccattaagacccttcgtaaagtggtgcgttctctccccaggcacggggagcgggtcctttatgcctccgccttctctcctgcagggctgtgtctggctgtccctgacaaaacggtcaaatggtgcgcagtgtcagagcacgagaataccaagtgcatcagcttccgtgaccacatgaagaccgtccttccgcctgatggcccccggcttgcctgtgtgaagaaaacctcctatccggattgcatcaaggccatttctgtaagtcctgctgccgggaacggtagcccctaTACGCCTCTGGTTTGACTCACTTGTGGAAAAAAAAAAATTGAACTGGGGGGTGGGGACTGTCAGGTGTCCAAGT
Deleted Sequence:
ccagggttaggtgggaacatggcagaccgttggggcgaacggattcatctgttcccaggatcggagtgcagcagagccctgtgctctgtcatgactgccaccagggatgagaggagggtggaacagaggagaggtggccattaagacccttcgtaaagtggtgcgttctctccccaggcacggggagcgggtcctttatgcctccgccttctctcctgcagggctgtgtctggctgtccctgacaaaacggtcaaatggtgcgcagtgtcagagcacgagaataccaagtgcatcagcttccgtgaccacatgaagaccgtccttccgcctgatggcccccggcttgcctgtgtgaagaaaacctcctatccggattgcatcaaggccatttctgtaagtcctgctgccgggaacggtagccccta
426 bp deletion beginning at Chromosome 9 negative strand position 103,228,289 bp and ending after 103,227,864 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37632 | TTC AGC TAC CCC AGC ATC TC | Common | A | |||
37633 | TCC TGG GAA CAG ATG AAT CC | Wild type Reverse | A | |||
37634 | ACT TGG ACA CCT GAC AGT CC | Mutant Reverse | A | |||
37635 | Fluorophore-1 | AGG TGG GAA CAT GGC AGA C | Quencher-1 | WT Probe | ||
37636 | Fluorophore-2 | CGC CTC TGG TTT GAC TCA CT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37632 | 0.40 uM |
37633 | 0.40 uM |
37634 | 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 |