Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:118647885+118647989 105bp TGTCACCCATGAAAAAGCTG TGGTGGAGGAGGACTTTGTG
Mut = 107 bp
Wt = 105 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AATCCCAGAAATGTGAGATTATGCCCCTATAGTGTATTAAGCAATAGTTGATGATCTTTTGTTGTAGAAACATGgatagtattctcataatctctctgtgaggttatattgttatccccatcatacagatgtggaaactgaggctcagaatacttaatggggcaagagccctctattggcagactttaggctttcaggctatgcctgtctgatcctgtcacctgtacaacattgtgctggtaattctcccttctttgagagaagaatttggactgcccatattacctttcattatcactgagtgacctgtctcatccctgcatgcctcaacaccttcataataatgtttgcacattttggtttctgccttcccccagtatggccaagtacttccggaacaacttcatcaacccccacatatactccagagggattgctaagctcatcttttgctgggacttcactgtcacccatgaaaaagctgtaaagctaaaacagaagaatctgagcacggagatcagggtaagtcccagactccagagtcctccccacaaagtcctcctccaccagccttcctcacccctgcgtgtctacagcttttttcctccccccccccctcctcttccctgcttttccttccctttcccaattccctttctctttccctttgtgatagggtttcagccaggctgaggtgtgtacatcttaatcccagcacctcaggagccaggggcaggtagatctttgagagttctaggctggggcgggggtgggtgggatggtggggtggagtggggtgaggggtgttcatgtgtatcacaagctgatctggaatttaagatcctccttcctcagacttcctggtgctgagattataaacctttgcctccactcgtggtcttgtctataaaatatttaattatggataacacccaatgccagccagtgcatggggacatcatgccaggctttgtggttgtctcagtcttcactggggccagtttagcaattcctctgaaaaaacatcctcttcttgagcttccacattaaaagttctaccctagagcagtgattttccatctggagcaaccgtgtccctcagtgcgggggagcttgggctatctctgaagaccgcgttcagttgctacagttagggtaaaggtgcggtggtgtctagtgggtaaggtttgggatgccatgaagcactccatgtacaaggcaagcacagtctgaggcggaactgccctggtctacagagacgcctttaatcccagcattggaggggcagatgctggaatccaaggccaacctgatctacacaatagggagttccaggacagccagggttacagaatgaaaccctgtttcaaaatacaaatcaaaacaacaataacagtaaagcccatcaaaataaaataaaccataacttatcaataaatgagctttgatggcagctttaacttcttccccccatgtggttctcagaattgttcgttaagaaaggcttctgtggagtgttttgttatggtatacacctgccatcctggagaggcggaggctggggattgtatgtttaaggacaggctaggctacactgtaagaccttgtttcctccaaacacaaagataaaaaagaagaaaggcaggaagagagtgtctgaaggagggagggagggagggaggagggggaagggaagggatgaagagagacaatgaggagaggaggagaagaggaaagagaaagaaggagcagagggaggcagcacagaagaacgcacaccatgaccaagggcctcttaggtctctctgttggtccaagatggaaatggagtttgaagtccttgtgcttttctggcgtcttgaggtttccccttggtctcccatgatccagctcgcagacttgcagcagacttaccctgcagcttctgtgtgatctgggctagccctgtgctcctgaggattgctgctgacaagcagggaagggccagtgggtcccactgactgttctcctggcacttgcaggagaacctgtctgagctccgccaggagaactacaggctcacattcaaccagcagctgacccgattttctgctcacgtagccgcttggctcgtctctactggagtgaccgcagcctgctgtgtagctgtctactacttggctgagtataactctgaggtaactctgcggaccagagcagagagagctggggaggactgggaggccactgggcagatgcagagcagatgcctccagcagggccctgtggccagcagcaactgcacccatagagaccttgcttcaaaatgcagaaccaagcctcttctcacggccagatctgcacttaggagcccccagggtgTAATGCACATCACAGGGCAAAGTGTACCAGGCTCCTGTGGACGTGGGACTTTGGCAGAGAGCTGG
This mutation is a 2287 bp deletion beginning at Chromosome 7 position 118,647,495 bp and ending after 118,649,781 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
53237 | TGT CAC CCA TGA AAA AGC TG | Wild type Forward | A | |||
53239 | TGG TGG AGG AGG ACT TTG TG | Wild type Reverse | A | |||
53240 | ATG CCC CTA TAG TGT ATT AAG CA | Mutant Forward | A | |||
53241 | CAA AGT CCC ACG TCC ACA G | Mutant Reverse | A | |||
53242 | Fluorophore-1 | CAC GGA GAT CAG GGT AAG TCC | Quencher-1 | WT Probe | ||
53244 | Fluorophore-2 | ATG TAA TGC ACA TCA CAG GGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
53237 | 0.40 uM |
53239 | 0.40 uM |
53240 | 0.40 uM |
53241 | 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 |