Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:108980478+108980563 86bp ACTGTGTCGATGTGGTCAGC CCTTTCTCCAGGGCTAAGGA
Mut = 84 bp
Wt = 86 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TCGTAAGCCGCTTGCAGGAGTCAAGTGTGTACTGGACATCCTCCCAGGAGCCTCATCCCCGTGGAGAgccaggatgagggggaggggaaggtaccccttgcttaagaagaggttaccaccgccccccccgccccccccagctccttgagctctgaggctggcccctttctgggtctcagtcaactatggtctgactgggtgctctctccattccaggacaggtagcagactgtcgaaggcaggggcaggactgtgtcgatgtggtcagcaggtaggagcgggtgtgaaggctgagtggagacaggagatgctgtttccttagccctggagaaaggagacctgtgctttgctgtgaggaagagagactctagctgccagttagatgtgaaattgtgggaccccaacctcctggggttccttcatcacggtgactggtgctgcctgtctttgtttcccttgcaggtgtggaatgctgtggactcgggacactgcctgcagacctactctgtgcacagtgaggcagtaagggctgcacggtggtctccctgtggccggcgcatcctcagtggtggcttcgactttgccctgcacctaacagaccttgaaacaggtgtgtcctaacgtcctaactgtctctggtcagccagcagccccctcactggggaagcctcctcagcgggtcctgactgcagactgtctatggtgacggtgtggcagaggcctcaggacatgcctacggGCATGCAGATCATTTGCCAGCAGCACCACAGCTGTCTACAAAGCATTGGAAGCAGTCCTCTGCCAGTCAGCAAAGGACAGAGCCAAG
This mutation is a 672 bp deletion beginning at Chromosome 12 position 108,980,296 bp and ending after 108,980,967 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48376 | GCT TGC AGG AGT CAA GTG TG | Mutant Forward | A | |||
50470 | ACT GTG TCG ATG TGG TCA GC | Wild type Forward | A | |||
50471 | CCT TTC TCC AGG GCT AAG GA | Wild type Reverse | A | |||
50472 | GTG CTG CTG GCA AAT GAT CT | Mutant Reverse | A | |||
50473 | Fluorophore-1 | AGC GGG TGT GAA GGC TGA GT | Quencher-1 | WT Probe | ||
50474 | Fluorophore-2 | CTC ATC CCC GTG GAG AGC AT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48376 | 0.40 uM |
50470 | 0.40 uM |
50471 | 0.40 uM |
50472 | 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 |