Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chrX:135843474+135843576 103bp GGAAGAGCAGGACTCTGTGG AAGATGCTCGCGAATTTCC
Mut = 108 bp
Wt = 103 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TGGGAAGGTACAATACTGTGACAGACCTGTCATTGAGACTGAGGCTTTGGTAGGCATTCTTAAATTGAGCCTTCTAACTTGTACCATGACTGGGGCAGAAGTTGagaccggttctcaggccaagcctgataagaagcctcaggaagaggtcgcaggtggggctgagagagaaagtgaagcccctctggtggtcagacccaaagtcaggcctcaggctccagcaacaagtggtgctaggcccaaaactgaaactaaatcttcctctcgggcaaggcctaaaactgaaacccagtcagtgtctggaacaagacaccgaatgagtggagcaaggccgagatctgaggctcaattaatgtctggggccaggcctaaaaccgatgccagggcagtaggtggtgctcgccctaagactgaggccaagccaatcccaggagcaaggcctaagggtgatgcccaggcttgggcccacagtgagtttggagccgaggcaatgccacgtgctgagagagcacacttatctaattctgtcacatggccaccagtcaatgttgggtctgcaactgttactaaatctaagagtctgtctatgaatacagaactggccagtatgggtagtgaaatcttttctggcacccagggtcagcctggaatcgagccctggtttggaccgagagaagaggctaatatgggttcttggtgctatcccaggcccagagccagagaggagacctctaatgagtctgcagatgaaaactctacaatgtcttctttctggactagagaggagactagtatcagatcatggcccagggaagaggtgaacactaggtccaggcacagggctaaacatcagactaatgccaggtcgaagcccagatccaaacaagatccctatattgattctttgtctgggtccgaggatgaggccagtaacccattctgcttttgggctggagaaaatactaatgacatgtttagggccagaggcagggatgaggcgaatgccagacccaagatcaggacaaagagagaggactattttgaagatgaagatgagatctataaagagtcctggcttttgcctggggaagagggtaatagatttaggcgccgagacaaggaagagcctaataagaccttgaaaaatgagaacgaaaaagatgttaaaaatgatgaaacagtcgaacaagagtccaggcttgaggaggaagtcattattgggtcctggttctgggcagaacaggagactaatgtggaggctggagcttcagcaatctgtgacgctgaaccaggggctgaagagggggccattggtggatccctgttctggaccgaagaaaagcctagtttgggggctgtggccagagatgaggtcaggcctgagtctgaagaagaggcactatttgggtcctggttttgggatagggatgaggcctgctttgacccaaatcctactcctgtgtacacagctaagagcaggtacagagatccagaagaggatcttaatttagcatccaggcccaaaacctgggacgaggttaccatcgaattcaaacctccttgccatgggcttgggttcccttccccaagaccctttatcattcctgaaggggcttctggaaatagtgaggaaaaagccaagaatgcagagcttggcgcagagggggaagagcaggactctgtggctcagcgtgatcttcctgaacccgagtttccatttcagtatgatccctcttaccgctcagtccaggaaattcgcgagcatcttaaggccagggaaagtgcacagccagagaattggtcttgcacctgcatccagtgtgagcttagaattagttctgcagaatttgaggagcttcttttactgatggacaggattcgtgatccttttatccatgagatagctaagattgcaatgggcatgagaactgcttctcagtttacccgggatttcattcgcgattcgggtgttgtctcactgattgaagccttgatgaattatccttcctcccgagtgaggactaattttttggaaaacatggttcacatggctcccccttatccaaatctaaacatgattgagacatttatttgtcaagtgtgtgaggagacactttcacatagcgtgaattcccctgaacagctaactggaatgaggatgcttagacatctcactataactactgactatcacgtactgattgccaattacgtgtctgggtttctcgccttattaaccacaggcgatgcaagaaccaagtttcatgttctgaaaatgctgttgaacttgtctgacaatccgatggtggcaaaaaaactattcagtgccaaagctctatcaatatttgtgggtctctttaacatagaggagactaatgataacattcagattgttattaaaatgtttcagaacatcagtaatattgtaaaaagtggagcgatgtctttacttgatgatgatttcagtcttgagccgcttgtttctgcatttcatgaatttgaagagctagctaaacagctgcagatccaaatagacaatcaaaatgatcccgaGGAGGGACAGTAAAGTTAGTGTGATTTAACCACCTGCCACTCATCAGCCTTATGTTCCCAAAGAGTCCCAAATAGTGCTTTGGTGTTC
This mutation is a 2449 bp deletion beginning at Chromosome X position 135,841,913 bp and ending after 135,844,361 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51466 | GGA AGA GCA GGA CTC TGT GG | Wild type Forward | A | |||
51467 | AAG ATG CTC GCG AAT TTC C | Wild type Reverse | A | |||
51468 | TGA GGC TTT GGT AGG CAT TC | Mutant Forward | A | |||
51469 | TGA GTG GCA GGT GGT TAA ATC | Mutant Reverse | A | |||
51470 | Fluorophore-1 | CTT CCT GAA CCC GAG TTT CC | Quencher-1 | WT Probe | ||
51471 | Fluorophore-2 | ACT GGG GCA GAA GTT GGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51466 | 0.40 uM |
51467 | 0.40 uM |
51468 | 0.40 uM |
51469 | 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 |