Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:117887454-117887547 94bp GGATCTGCAGTGATTGGTGA CGTTCTCCGTGACAGGTACT
Mut= 87 bp
Wt= 94 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TATAAGATCACCTGGACATCGTGGGCGTTCGCGTCTGGCATTCCACCCTACCTCTGGGTTGGAAAAGACAACCTAGAATGACCTCCGATGAACAGCAGGCATTAGCTAGGCACCGCGAAATCCTGCTTCAAGCAGAAGGAACTAGGCAGGACTAGAACAGACCGGAAGGATCTGCAGTGATTGGTGAGTAAACTGGGAGTCcggtgggaagttagggaaccagcagcgcaggtggagagccagtacctgtcacggagaacgtccgacgaaactacaaccaccacagtgctccgcggcatgacgtctaccagggcccggcctcaggcagccagccactgtgcgcgggggtcacgtggggtcggcgcatcacgtgcgccggtcaggtggcgctgcggggcgggccgacagactgcggggcggacggtggacgcggtttagctcaggtcccaccgatccgacccctgccgtcgccgctgccatgacggggctggagttgctctacctcgggatctttgtggccttctgggcctgcatggtcgttgtgggtgagcggggcccaggcggcccaaggtggcctgctggttggggcggggggcaggtggcagccggagccggccccgcgcggtgccctcccgcgacggaggaggccggccctgggctgcgagagtcacaccggctattccagggtcgcgacgccaggttcctgacctcctctccgcgagccgcatatttgggcgccccgcttcccctttttcctccttgccctcccccacccattcacagcttgttgggtaaacagcgattgggccggcaggatctggcgcggcctggacgcagcgcccgttccctccggcccgtgaaggggcttggtgtcattaagacgggttacctagggagtggccgaaaaaaaaaaaaaaatggaggcctgggtggaggtgctgtttcgctccgggactcttcgcttgtctagatgtaggtggctgacagtttgacagttgggcagttttttttttttcctaggttggggactgtgtctcgaaagagcaaccctttaactcctttcctcattgcttcaggaatctgctacaccatctttgacctgggctttcgctttgatgtggcatggtaagggagggcatggagaggattctcctaggagcctcttctccctgctttggtctgagccccagcccagctcttcagacacacggacctttgggaaatagaaaatctagagatgcaacgtctgggactggtgggctggagctgggtggacactgagttttgtgtggcctccccactcctgccctttcctgtccatagctcaatagctttcttccctgtttccacaccaggttcctgacggaaacttcccccttcatgtggtcgaatctgggcattggcctagcaatttctctgtctgtggttggagcagcctggtgagtgttggagtggcgggacgagtgggaggtgccaagtcgtggcaggtggaatcatcactgggttcttaatcatcctggggacagcaactccttgggctcctttaatcagtttcttacccttcaccaggggaatctatataaccggctcatccattattgggggtggggtgaaagcccccagaatcaaaaccaagaacttggttaggtaagtgtaaaggcttctgcatgtcaaaactagctgcgatgggggtcgggtgacatgctggcttgtgacccgtctcatcgtagcgtgtatgtgagtcagggaaagttggctggggattttctgcagaaatggctgatttccttcttaaagagtttccctccccctcccccaccatttaccatcactgttgccagtattatcttctgtgaagcggtggccatctatggcatcatcatggcaattgtcatcagcaacatggctgaggtaagggaaaaccagcggggacgggtatccattgaaatgcctatgctttccctaaagatcggagggaagaaaaggtaggtgcattaaggaatgccttctgaggacttattctgcccctcacagcctttcagtgctacggagcccaaggccattggccatcgaaactaccatgcaggtaagtgggtaacatagagctcactcggcatggtgctctgggcctttaaccccagtcctgggacgggggtagcaccaagtgagttaagaggccagccagcactacataatgacatagtgagacagacaccctaccacacatacacgcctgggccttacacacttgcttctaccacacgtacacacctgggccttacacacttactcctgcttcctggctccatagctgagagtctcagctctgcagagttggggtgggactggccctgctagcccacactggaccattcctctcctgcttgcctccacctcacgagctacttcttatcctccaggttactccatgtttggggctggcctcacagtcggtctgtccaacctgttctgtggagtctgcgtgggcatcgtgggcagtggggccgccctggcggatgcacagaaccccagcctctttgtaaaaattctcatcgtggagatctttggcagtgccattggcctctttggggtcattgttgcaatccttcaggtgcgagtccccctagagaggaacagaagtgcctcccttttttccccttcccatctcttggtccccctttgtctcctgctctagatgcacgcatctttcctaagtctctccattagcatctctttcctcggctgcctctgaaaatgtctttgtggagtttctgtctctcagggtctgtgttctgtatgtatgacacacttgtcagtagactagcatgtctaaattcatattttccagtgtgtgagtttgtgacttgggtgcttgactgggctgactcttggcgacactaacactgtccatgggacttgtcttctctgttgtacagctgagctcctttgttactgtccatgtctgtttgtctgtcatagtgtgtctgtccgtctgtccacccactggcctgctggttctggtcttgtcactggtcactgtccctgcctgatttgtcctgtttttcctttgcagacctccagagtgaagatgggtgactagatgacctgtatgggtggggccgtgccctactgttatttattgctggttttcctgggacagctggagctgtgtccctcagcctttctggggcttggtgttcagggtctcccctgcactcacctcttgctgcctgttgactctggaggcattgcagtccagactgaaccccagtgtggggagtgggctgctgatggtgactgtgtacagctgtgcacctgtgcccccctcaacccccatccagcccatcttcctagtgtttgtgaaataaacttggtatttgtcccagtcagtgaggcttctgttggccccttgcgacacctttcacaaaggaaggggaggacctgttcactcactccaacctcctgtaactttctgtctacacatcatctctgctctggaccttgctggctcctgggtggcagatcagggtgctaggctggctgtaatgctggcacctcttcattgaagagtctgggctctgagatcctcctagccctatgtctttggacccctgtgcactagacccAAACTCACTACAGCTAATTCTCCTCTCTGTCGCTATACTCCTGAAACTCCCCGCACCTGGATTTTCCTGGAGAACAGTGTTTTACATTTGATAGTCCAGTATGAACACCTAACCCTTCCAGAAACCTCTTCTATGGTCTTAGTTCCTAGGGCCTTGTTCAGCTCCGCCTATTCCTATACAATTCTTGATTCCAACACCA
This mutation is a 3425 bp deletion beginning at Chromosome 4 position 117,884,089 bp and ending after 117,887,513 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52624 | GGA TCT GCA GTG ATT GGT GA | Common | A | |||
52625 | CGT TCT CCG TGA CAG GTA CT | Wild type Reverse | A | |||
52626 | CGG GGA GTT TCA GGA GTA TAG | Mutant Reverse | A | |||
52627 | Fluorophore-1 | AGC AGC GCA GGT GGA G | Quencher-1 | WT Probe | ||
52628 | Fluorophore-2 | CTG GGA GTC AAA CTC ACT ACA GC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52624 | 0.40 uM |
52625 | 0.40 uM |
52626 | 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 |