Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chrX:122396919-122397029 111bp GGCTAACAGTATGGCAGAAGC TCAGATTCATCCCCAGAATCA
Mut = 114 bp
Wt = 111 bp
Fam = Mut
Hex = Wt
X-Linked
Wt Sequence (deletions in lower case):
GTCCTTTTCTGTCCGTCTCTGGGTGCACGATCTCTGCTGTTGTCACCACCTTAAATAGGTGTACGGGGCTAACAGTATGGCAGAAGCGGATCCTAAAATGGTCACAGAACCTGGTGCCcatggggttgctgaagaggcgatggctagcacagcttgtgattctggggatgaatctgacagcaatagctctagcagtaccaatagttgcagcagcagcggcagcagcagcagcggcagcagcagtagcagcagcagcagtagcagcagcagcagcagcagcagcagtagcagtagcggtagcagtggcagcagcagcaatggcagtcatttgaaccgaaagaagagggtacctgagccttccagaagggcccagcgacgtccctccgggaaacttttcttggataagctgccccaagccgtaagaaatcgggtgcaggcactcagaaatattcagaatgagtgtgacaaggtagacaccttgttcttaagggcaattcatgatcttgaaagaaagtatgctgaactcaataagcctctatatgataagcgttttcagatcataaatgcagaatatgagcctacagaggaagaatgtgaatggaattcagaagaagagttcagtggtgatgaagaaatgcaggatgacacacctaatgaaatgccacccttagagggtgaggaggaagaagaaagctgtaatgaaaaagctgaagtgaaggaagaaggaacacatgttccagaagaagttcctgaggcaaaagttgaagaagaggaggctcccaaagaaactcctgaggtgaaaactgaagaaaaagacattccaaaagaaggtgctgaagaaaaagctgaagaacaggaatcctctaaagaaattcctgaggtaaaaggtgaagaaaaagcagactctacggattgtatagatatagctcctgaagaaaaagaagacgtcaaagaagttacccaggcaaatacagaaaataaggatcaacctacagaagaatttacaccaagggctccagcaagagaggctcaaaaaagggtccctgagacaaggcctgaagaaggagtcaatattaaaagggctcgaaagggaaaacctaagaaagaagatcctaaaggtattcctgactactggctgactgttttaaagaatgttgataagcttgggcctatgattcaaaagtgtgatgaacccattttgaagttcttatctgatgtgagcctgaagttctcaaaccctggccagcctattggttacacttttgaatttcatttcctacctaacccatacttcagaaatgagctcctgatgaagacatacataataaggtcaaaaccagatcactacgacccgttcttcgcgtggggatgggaaattgaagagtgtaaaggctgcaaaatagactggagacgaggaaaagatgttacggtgacaaccacccggagtcgccctggtattacaggggaaattgaagtccagccaagagtggttcctaatgcatccttcttcaatttcttcagtcctcctgagattcctttgattgggaagctggaaccaagagaagatgctatccttgatgaggactttgagattggtcaaattttgcatgataatgtcatcttgaagtcaatctattacttcacaggagaaatcaatgatccctactACCATGACTTCAGGGATTATGGAAATAGGAAGTACTACAAGTAGAAAGGGCAAGCTGAAAAATCTTTCAAGAATCCTTAAAAGTTAAGCAAGCAGAAGTGAAATAAGAGTCATCTAAACTTCTAAAATAGAAAACTTGCCCCATAACCTGAACAGTATTACTCCTTATAGTC
This mutation is a 1549 bp deletion beginning at Chromosome X position 122,395,429 bp and ending after 122,396,977 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51259 | GGC TAA CAG TAT GGC AGA AGC | Common | A | |||
51260 | TCA GAT TCA TCC CCA GAA TCA | Wild type Reverse | A | |||
51261 | TTT TTC AGC TTG CCC TTT CTA C | Mutant Reverse | A | |||
51262 | Fluorophore-1 | CTG AAG AGG CGA TGG CTA GC | Quencher-1 | WT Probe | ||
51263 | Fluorophore-2 | CCA CCA TGA CTT CAG GGA TTA TG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51259 | 0.40 uM |
51260 | 0.40 uM |
51261 | 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 |