Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chrX:157818305+157818392 88bp AAGCTGAGCAGGCAAAAATC GCTGGAGGCATCCCATAAC
Mut = 118 bp
Wt = 88 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AGCCAAATCTGGATGAAGGAACCTTAGGTTTTAAGCTGAGCAGGCAAAAATCAGCCTTTGCTAGGGGCTCAGCAGCTGgacagcggaggtgggggttccgtgttatgggatgcctccagccgcgctgggctggctccatcccagcttcccgccttgggcatcatgagttacttcctgtcttactgcaaaacccacggcagctctcttctcactggctaccaggccctacgcgctgagggcttcttgtgcgacgtgacactggaggccgagggcagtgagttcccagcacacaggtcacttctggcgtgctccagcgactacttcagggccctgttcaagagtcacactcaggaatcgcgggctcgcgtaattcacctgcgtgtgccttcagcggccggcctacagcgcctgctggacttcatctacactgcctggctgcccctctccatggacactgtggaggacactctggaggctgccagctacctgcaagtcaccgaggccttgggactgtgtgcccactacttggaacgccagcttgccccagagaattgctgtttcattgccaatgtggcagctcgatttggcctggcccgcaccctaggtgcagcggagtgctgtattgtacgccacctggggggcctgttactgatgggcactggcccctcggggctgttggaactcaatcctatatcaatgaaagctgtgctgggcgcccctgacgtggcacgggtgcctgagacgcggctgctgggcctggcactagcctggctgcagcaggaacccaaggtccagcgcctggcacatagttcaacactgcttggatgcatccgctttggcctggtgcctgctggtgtgctgcggcgcatatactcgggctccggtcttatattacctactaggatcaaaagcctcatcatccaggccctcaattaccatacagcaccctccagccagccgcttctgcagggagaacagactagtgttcggagtccccagacccgtatcttgctagttggtggtctcagagctagggagatggttaccgaggagatcatgggcctaccagaggtagttcagaatcggcaccctgcacctcaaccagaggaagaggagggacaggaggaggaagagatggaggagaaggagtgggagctcacccaggatgtggtgtccttcgacgtgtataaccacagctggcacagcttgacacagctgcccacaccactgctaggccacagtgtatgtaccactggcaacttcctatttgtccttggaggggagagccttcctggcagcccatctagttccctggctgccggctcaaggtcaatcacagccttagtgcaccgttatgacccgcgcttccacacttggacacaggtgcctgccatgcgagaagcaagggcctacttctggtgtggtgtggtaggtgagagtctcctggctgtcggaggcctgggcatctgtggtgaggcactggcctcagtggagatgtatgaccttcgcagggaccgctggatggcagctggacagctacctcgggcagtacatggtcacgcaggtgctgtcggggatcatggtgttgtgtacatctccgggggcaaggctgggagaggtgaaggaggcacaagtagcctccgggatatgtactcccttagccccggagagagagtgtggagcaagagggcacccatgggcacagctcgctatgggcactaccttgtggtgctgcGAGGTGCAGTGTTTGCCTTTTTGGGGAGATATGAGCCCTTCTCTGAGATTGAACGCTATGACCCCTGCACAGACCAGTGGACTCGGTTGCGGTCGCTACCCTATGACCGCTTCTGCTATGGA
This mutation is a 1657 bp deletion beginning at Chromosome X position 157,818,351 bp and ending after 157,820,007 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50297 | AAG CTG AGC AGG CAA AAA TC | Common | A | |||
50298 | GCT GGA GGC ATC CCA TAA C | Wild type Reverse | A | |||
50299 | AGG GGT CAT AGC GTT CAA TC | Mutant Reverse | A | |||
50300 | Fluorophore-1 | CAG CGG AGG TGG GGG T | Quencher-1 | WT Probe | ||
50301 | Fluorophore-2 | AGC TGT CCA GCG AGG TGC A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50297 | 0.40 uM |
50298 | 0.40 uM |
50299 | 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 |