Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 74 bp
Wild Type = 88 bp
Wt sequence with deletion in lowercase
GGTTGGAATCTGAGTAGTGCATAACAGCATTAGGGTACATGCCTGAGAGGGACAGAGGAATCAGCTTCAAGGTCATTCCTGTATAAGCATCAAGTTTGAGAGCAACTTGGGACACACACACAGGTCCCTATCTCAGAGAGGAAAAAAAATCGCAAGCTTGCCTGTGGCCGAGGACGGGCGTGCATGTGTGCAGTTTCCTCTCTGAACCTTAGGTTTTGGTCCAGAagttggaagtgtgggaagaacggggaaaattgagggaaagggaccgaccaggcaactcctggaggcggtggggtgtgaggggctgtggctgccccgtctctcacttgtgcctctgtccctgcaggtgagtcagtggccagctctatgcattcctcgcgctaccctagcccagcggagctggatgcctacgcggagaaggtagccaacagtcccctgtccatcaagatcttccccaccaacatccgagtgccgcagcacaagcacctgagccgcacggtcaatggctacgacaccagcggccagcgttacagcccctaccctcagcacaccgccggctaccagggccttctggccatcgtcaaggccgccgtctcctcgagtgctcatgcgcccgccggccaccccaaaagcgtgctcaagagtgtggagggcaagcggactaaactgtcgccggccaccgtgcaggtgggcattgcgccttacccagtgcccagcaccctggggcccttggcctacccgaagccacccgaggcacctgcaccaccgcccagcctgcctgcagcagccactgccacctctgtgatccctctgcccggccggggccttcccctccccccctccaacctaccctctatccatagcatcctgtaccagctcaaccagcaatgccaggccccgggtgctgcaccaagcgcctgtcagggcgtggctgtgccccaccccagtccagctaagcatggcccagtccccagctttcccaacctggcttactcggccacagccggtctgccagactgccggaagggcacagaactgagccagggagccaccccagccctgacactggctggggccaccaaacctgcagggtatgcagaaggtggcctggattacctgctgtggccacagaagccacccccacccccaccccagccgctgcgggcctacagtagcagcaccgtagccggcaagtcccagtccccagagatttgtggcgggagggccttcgaaagggccaacgggtcgccccacaactgcggaatggggctgcccggcagcttcaccgtgggccagtacttcgctgccccttggaacagtgttctggtgacacctaccagtgactgctacaatccagcagctgctgccgtagtgacagagctggcaccaggagccgcccgggagctagccgggcctcctggagacgtactctcgggcctgaccagcaagagtgtgtgtaacacagcggtgctgagcagcagcctccagtcgctggagtatctcatcaacgacatccggccaccctgcatcaaagaacagatgctgggtaagggttacgagacggtggccgtgccccggctgctggaccaccagcacgcccacatccgcctacctgtctacagataaggtctgccctgcggacacacggacagggtgatgggggttgccaggccatagcacagggcgggaagctagcagggcgccccgcccgccctgtgtccacggggaagccaggctggttacctcctcacttcagtgggcaggcgggttgacctcacacaccagggcttctttcctctcccgcttgctgccccaggcagcagggagggtcccaggctggccacgaggctccttcatctttcatccaggcagggaaagaggaccttctgccaacaagaagttccttctgggtctccagcagagggcgctccagtcatcccgtgtcatacacggggtctctatccacaatctcccagtccctgggtccaaagtgccccaccccaccaactaatccaggtgaagaccgccctgactccacacccccagaggtctcaggactaccccccccccgttcctgctgactctccccctacccacccaccctccctactaggaatcacgcccaacctacccggtacgccccagaccaacctcttctccatccctacactacatctcacagggctgctgggaacagttggtgcgttgcacaagggcaccttgcccaaggagactccacacacatcctagatttgtgtatttattacagttgtctgcatttcaaggaagggacactatcttcctattcgcacaaagatgtcacggggggctaacaagggacctgtaaattcaggctccattcacaaggacggtcttgccttcttcgcatctctgggtctcctggggaccagtgggagggtgacctggctgtaactggaatacccaatccagctcccgttctcattccccccacccccaccgcactgctgcggggcgtggggaagagagaagcccaggaagtcctcaaggcctgcagtccccgggccctgccggccccaggcgctccggaaagcggtactgtatctcgccaaggcctgtctgagcactaagtgcatttacaaagctctgagaatgttttttttatactaaaattgaccattatattctactgtgagaagtgcagtctgtctgcactatattgttttaaaaacgaagagaaagaaaaaaaacggaaaacagatggtgtctcagctgtgggtttgctgtaaggctgttctctctctctccctggcttcccccacctcagggatccacgggagggcaccccaaggagccctgtggggacagagtggggtggcatgcagggtgagctatgcccaacttattacaaagctcttcaagataaatgctatcccgtgtctatgtgtcaggcccgtggTCCATCCATAGGGGCAAAATCAATCCTGAGCCCAAGCCCTGGTGGCTCTTTTCGGTGCTGAGGTGGCTCTGAAAACTCCCCATTGGAAGTCCATCTCTAGATCCTGTTGTCATCCTCCCAGATTAAATGGTTGCCATGTAGCTGAGAACAGGCTAGCCATCGCCCTGGCCGGCATGGATTATGCAGGCTGACACCTGGCATATACCCATACCC
This mutation is a 2716 bp deletion beginning at Chromosome 5 position 114,610,703 bp and ending after 114,613,418 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44244 | TGC AGT TTC CTC TCT GAA CCT | Mutant Forward | A | |||
44245 | GCT TGG GCT CAG GAT TGA | Mutant Reverse | A | |||
44246 | CCA CCA ACT AAT CCA GGT GA | Wild type Forward | A | |||
44247 | GGG AGA GTC AGC AGG AAC G | Wild type Reverse | A | |||
44248 | Fluorophore-1 | CCA GAT CCA TCC ATA GGG GC | Quencher-1 | MUT Probe | ||
44249 | Fluorophore-2 | CCG CCC TGA CTC CAC ACC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44244 | 0.40 uM |
44245 | 0.40 uM |
44246 | 0.40 uM |
44247 | 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 |