Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chrX:126815418-126815523 106bp GAGTATATTTGAAGAGGTTTCTGACTC TTCATTCTGAAGGCAAAGCA
Mut = 119 bp
Wt = 106 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AAGTTGAGTATATTTGAAGAGGTTTCTGACTCAGTAACAGTTCAAATTATGAGCTCTGTCTTacttagtgaggactcagacaggaacctcatgctttgccttcagaatgaagctaatgaaatggaaaatcctactgaacctctgaataagaggccttgtttgtcctctgaagttgattctacttcgtatatgtcagctcttgaattcccactacaagaaatggagcaaagttttggtttggtcacaaccacagccacagaggtggctgaggaagagtttggtggggaaaatatgatgcaattcgaggttttaaaagatgacaaacaatatgaagttttccccttgcctaacaaggaagagtcagtagttagccaagcatggttcacaagtaaggaagctaaggatgctctaactcatcaaggacagaaatggaagcaggggatgtggtccaaggaagaaacagctattctgatgaacaacattgaaaggtatatgaaggaccatggagtagagaatcctgcagaaatcatttttaagatggcaaaaggtaaaagaaaagatttctacaggtctgtatcattgggtctgaatagacctttgttttcagtttatagaagagtggtccgaatgtatgatgacagaaaccatgtgggtaagtacagccctgaggaaatagagaagctcaaggagctctggcagaagcatggcaatgactggataacaataggggccgccatgggaagaagtccatcttcggtcaaagaccggtgccgactgatgaaggatacttgcaacacggggaaatggaccgaagaagaagaacagcttcttggcgatgtggttcatgaattgacatgcactgaggtggatgagaaagttacccatggtgtgtgttgggcaacagtggctcaacgagtgggtactcgctcagcaaagcagtgtcgtgctaaatggctcaactacttgaactggaaacagactggtggtattgagtggacaaggaaagatgaagtcactctcatccaaaggctagtagagcttgatgtatccgatgaaagtgaaattcgctgggatgaattagctaaaggatgggaaagtgttcgttcaccacaatggctgcgaaataaatggtggatcatcaaaaggcaaattacaaatcataaagattttgcattcccagtcctagtaagatgtcttcaacaagaatatgaaagccaaaatgccagcctcaggttttgggagaataaatcaggatctgaagtaccagatagcaaccctgatatcatttttcaacaagtcccctttggagtcacaagtatagaaaataataatgcatccatctgtactgaccccatgacaggattgcaaatttcattccagctcatccccagacccacaacagaattacctgatgctgttgttgactctgaaaccataaccctgcccaCTGGACCACAGTAGATATGTGAGAATCTCCCTTCTTTCCTTATGTGTTCCACTCCCTTACCTTAAACCTATTTCCTTCA
This mutation is a 1381 bp deletion beginning at Chromosome X position 126,814,082 bp and ending after 126,815,466 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52313 | GAG TAT ATT TGA AGA GGT TTC TGA CTC | Common | A | |||
52314 | TTC ATT CTG AAG GCA AAG CA | Wild type Reverse | A | |||
52315 | AAG GGA GTG GAA CAC ATA AGG A | Mutant Reverse | A | |||
52316 | Fluorophore-1 | CTT AGT GAG GAC TCA GAC AGG AAC C | Quencher-1 | WT Probe | ||
52317 | Fluorophore-2 | CTT TGG TCT GGA CCA CAG TAG ATA TG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52313 | 0.40 uM |
52314 | 0.40 uM |
52315 | 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 |