Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr15:85132604+85132707 104bp GGTGGGATCTACACCAGCAC TTCCTATATACACACACTCCCAACA
Mutant= 120 bp
Wild Type = 104 bp
Wt Sequence: CTTGCAGTAGTGAAATAGGCAGACTTTAAAACACAGAAAATGTGGTGGGATCTACACCAGCACTGACTGGAGCCTGGAGGGGAAATCAgagagggcttcacagagtagtggtacttaaacaatgttgggagtgtgtgtatataggaaatgaaggaagctctggtgacagctgacagcgcaggcagaagacccagccataagcaaaggcttccacgtatgggaagggaacctttaatgctgtggccagatgagagggttcgtggcctactgaataatgctagctggctctaacccctcttcccttgtccattcctagggagacacacaagcctgggattttcaagtccgtgaccagaagataaaagaaataactgacaaagctaggcatgaagcctttggtgagcacttctaaaatgcccagctatttcttacgGCCTCTAAAATATCAGCTATCTCTTAGGGCCTCATGTCTTCCTCTTACAGCTGAGAAGAACAGACACACACACACACACACACACACACACACACACACACACGT
Deleted Sequence: gagagggcttcacagagtagtggtacttaaacaatgttgggagtgtgtgtatataggaaatgaaggaagctctggtgacagctgacagcgcaggcagaagacccagccataagcaaaggcttccacgtatgggaagggaacctttaatgctgtggccagatgagagggttcgtggcctactgaataatgctagctggctctaacccctcttcccttgtccattcctagggagacacacaagcctgggattttcaagtccgtgaccagaagataaaagaaataactgacaaagctaggcatgaagcctttggtgagcacttctaaaatgcccagctatttcttacg
345 bp deletion beginning at Chromosome 15 positive strand position 85,132,649 bp and ending after 85,132,993 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37483 | GGT GGG ATC TAC ACC AGC AC | Common | A | |||
37484 | TTC CTA TAT ACA CAC ACT CCC AAC A | Wild type Reverse | A | |||
37485 | TGT GTG TGT GTC TGT TCT TCT CA | Mutant Reverse | A | |||
37486 | Fluorophore-1 | AGG GCC TCA TGT CTT CCT CT | Quencher-1 | MUT Probe | ||
37487 | Fluorophore-2 | AGA GGG CTT CAC AGA GTA GTG GT | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37483 | 0.40 uM |
37484 | 0.40 uM |
37485 | 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 |