Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mut = 89 bp
Wt = 98 bp
Fam = Mut
Hex = Wt
>chr7:112554848+112554945 98bp AGGACTCAGATACTGGGCTGAG GCTTGCTCCCTACTACTTCTACA
WT Sequence (deletions in lower case):
TCGGGAGCTGCCAGGTTGCCCGCGCTGGCCCTGCTGCCCTAATTGCACCTAGCCGGACTTGCTCCTCACGTCCCGGTTGCGCCATGgatccttcggagaagaagatatcggtgtggatctgccaggaggagaagctggtgtccggcctctcccgccgcaccacctgctcggacgtggtgcgggtgcttttggaggacggctgccggcggcgctgcaggcagcggcggggccagcgacggggcttgacggaagacccttcgggccagttggagctgcccgagcccccggacgaaaacgacgaggatgacgacgacgcgatgcccccgggcatgctatgtgggcccccgcagtgctattgcattgtggagaagtggcgcggctttgagcgcatcctgcccaacaagacgcggatcttgcgcctctggactgcttggggcgacgagcaggagaatgtacgcttcgtgctggtgcgcagcgaagcgtcgctgcccaacgccggaccgcgcagcgccgaggcgcgggtagtgctcagccgcgagcgcccctgcttggcccggggcgccccagcacggcccagcctggccttgacccaggagaagcagcggcgggtggtacgcaaggccttccgcaaactggccaagctcaaccggaggcgccagcagcagccgtcgtcaccttgttcgtccacgtcgtcgtccaccgcctcgtcctgctcgtcgtctgctcggacccacgagagcgcgtcggtagaacgtatggagacgctggtgcatctggtgctgtcgcaggaccacacgattcgccagcaggtgcagcggctccgggagctggaccgcgagatagaccgctacgaagccaaggtgcacctagaccgcatgcggaggcacggagtcaactacgtgcaggatacctatctagtgggggccggcatcgacctggacggacaaactcctgaaggggagccggaagatgcgacactggaggagaaggggacagaaccggcggcacccctggacagcgaggcgcaggcggcagcgttggaggagctggccaggcgctgtgacgacctggtgcggctgcaggaggagcgcgcccagcaggaggagttgctagagcgcctgtccgccgagatccaggaggaactgaaccagcgctggatgcagcggcgcaatgaggagctggcggctcgagaggagtccctagagcccgatggtggtccggatggcgagctgctgctggagcaggagcgggtcaggacgcagctcagcaccagcctttacatcgggctgaggctcagcacggacctggaggccgtcaaggcggacttggattacagccagcagcagagggacattaaggagcgcgagcttcagggccttctccagagtttgcacacttttgagcagacggtggtgcatgatggggctctgggttccagcggaccctctcgcgaacctcagcctcagacctgtgcagaaatgtgggtagaccaggcccggggactggctaagagttgcccgggcaacgatgaggactcagatactgggctgagctccatgcatagccaagactcagactcggtaccccctgtgtgtgAATCCCTTGTGTAGAAGTAGTAGGGAGCAAGCTTAGACGCTTGCAGAATGCACTGGTTTGGCCTGCTCAGG
This mutation is a 1510 bp deletion beginning at Chromosome 7 position 112,553,404 bp and ending after 112,554,913 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
58137 | GCT TGC TCC CTA CTA CTT CTA CA | Common | A | |||
58138 | AGG ACT CAG ATA CTG GGC TGA G | Wild type Forward | A | |||
58139 | CCT GCT GCC CTA ATT GCA C | Mutant Forward | A | |||
58140 | Fluorophore-1 | AAG ACT CAG ACT CGG TAC CCC CT | Quencher-1 | WT Probe | ||
58141 | Fluorophore-2 | CCG GTT GCG CCA TGA A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
58137 | 0.40 uM |
58138 | 0.40 uM |
58139 | 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 |