Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:139256340+139256442 103bp AACCGTAAGAAGAAGGGGATG TTTCAAACTGGGTCAAGAGC
Mut = 101 bp
Wt = 103 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AGGTGACAGGTGCTGGGCAGAGCTAGAGTTCTCCCTTATTCTCACTCTGACTCTGTTCCCTTCTACCAGGTCTGGCctctttggcctgtctccatctactctactgcctctggctgacaactcctctgccgtcagctgtcatggccaggctcccgaggagggaactggagaagtgatgcagctagagacaggacctctgcatcctcaccacaaagaccctgagaaggatcagcctcccaagacagctgtgtctgagccgcccccacctctcataccacctgtgcctgccgggaacctgcctccctttccaccatactttgagggtgcccccttccctcacccactatggctgaggaacacctaccagcagtgggtgccgcagcccccacccaggaccatcaagcggacccgtcggcgactctccagaaaccgggatcctggaagactcattcttagcacaatccgcctgcggccacgtcaggtactgtgtgagaagtgcaagagtactgtgagtcctcaagaggccagtcccagccccctgaacacccccaaaccccgccggaggctaggcagtggccctgacagtgaacaccgaaagccagaggaaccagaagacagcgctgtcatagctacagctgccccgaggaggagcaagagggagaagcgggaagaggacagggttgctggagagcgtgttccgagaagcccagtcatcaagatctcctacagcacaccacagggtaagggtgaggtggtcaagattccatccagagtgcatggctccgtggagcccttctgcccgcaacagtctctacagaatggcagccaggactcagaggtatcaagggatgtggagcccaggggtggtggagaccgaccacccagcgggtcttcagcctccatccccaagctaaagctgactcgcccagtgcctcctatctctgacctgccacctcccaaaatccgcctgaagccccaccggctgggggatggagagcatgaacccctttaccgggctgagctggtggaggagctgaacggatgcccacgaggcccgctggtcagctcccctgctctctttgccgatggctctagccatgggctagaggacttgtcttctggaagttctggtgaggacgatgacctcaagaggtttccccaaggtaaacatggccgtgatggcttggcttttcttgtggactgccctgggaggagaacagattgtaccagtgaatctgtgtgcagcacggacagcctggatgaattaaaatcatctggttcagaagtgacatctccagacacaggagacctgtcatctggtgacagtgcctctgttccttcatcttctgctgacactcgccagacagtccctcccctcacggtcaggctgcacacgcagagcgtctcacggtgtgtgactgaagatggaaggacggtggctgtaggggacattgtgtggggtaagattcatggttttccttggtggccagcgcgtgtccttgacatcagccttggccagaaggaggatggagagccctcttggcaagaagcgaaagtctcatggtttgggtctccaactacatcattcttgtctatttcaaaactgtcccctttctctgaatttttcaaactgagatttaaccgtaagaagaaggggatgtatcggaaagctataactgaggctgcaaatgccacacaacacgtggcCCCAGAAATAAGGGAGCTCTTGACCCAGTTTGAAATGTAACTTTGGTTCCAAGAGC
This mutation is a 1631 bp deletion beginning at Chromosome 7 position 139,254,777 bp and ending after 139,256,407 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51456 | TTT CAA ACT GGG TCA AGA GC | Common | A | |||
51457 | AAC CGT AAG AAG AAG GGG ATG | Wild type Forward | A | |||
51458 | TGC TGG GCA GAG CTA GAG T | Mutant Forward | A | |||
51459 | Fluorophore-1 | CGG AAA GCT ATA ACT GAG GCT G | Quencher-1 | WT Probe | ||
51460 | Fluorophore-2 | TAC CAG GTC TGG CCC CA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51456 | 0.40 uM |
51457 | 0.40 uM |
51458 | 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 |