Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:46997246-46997347 102bp AGTGGAAAAGAGGCTTATCTAGGAC ACCAGCTGCGTCCAGTCA
Mut= 104 bp
Wt= 102 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GAGACTTAGCTCTGTTTGTCATGCTTCTTTAATAGGTATTTAAGAGATTGAAGTCTTTCTTTCAGGGTTCAAATCATCCTTATTCGTGTTTCAGTCACTTTCTTTACGCAGTTACTTAAAAACCAAAGTCCACACAAATAGCATCTGTGCCCGGGCAGGATTCATGACCTTGGGATCTCGGAGAGCAGtgtaaaggagcttgtgtagcaaattaatagttaatgaacttatatgccgcagttgaagagtccagagagctgtgctctttccttagattccttcccttcctcttccttcctttcctcgctttgttttctgtgttttctgcagtactggctggggattgaactcctagctgataaatgtcagacaagcactcttcctctgagcctcacatcccagcccctagactatatatttgtaaagtgttgctgcttatagcttttattctgcttttgtttttagaaaaggtacagtttggcggtggggcctccaaagaaagacccaaaagtgaaaggtgtccagtcggcagctgtgcaagctttccttaggcggaaagaagaggagcttagacaaaaaggtatgcgcttgtactgcatctcagcctgtctgccaccaaatccgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtaaacatcctgacagcttagtagtttggggatgttgtatgtgtgtctgggttaactttatagacaaatatgtgcgtgcatgtttgtaaatgtgctcataggttggttatttgggttgttttttctaaccccatctcacctacccccaaggctttgtggagctggccacagtgatattcacctgtcatcccaggtattcaacaggctgagacaagatcatttattgagctcaggagcttcagagtagctgggacaacgcatcaaggccttatctcttgaaggaaggtggggagacatgaaaaggcctggtgtatcaaccatttaactagccataatatgtctccaattctttaccttggtcctggcattgttatagatattcattgattcactcttaacagctccagtcagtgctggtgtcctattgacatggtatatttatgagccattttgtgcattttaagcaaaagccaagtcttttatctgcttagtcataaagagaagctcatatctgacccacttgtgtagtaattactcagttttgttagatgacttcctcatttgacacagctctctagaaaccataactcagattgagctttgactgtggagtggcagcacatgagcgctgctatgctgacactgttgaggtgatgtgctcttgttttattcctttcagccttggaagagaaaaaaaggaaagaggaactagtgaaaaagcgaattgagctcaaacatgacaaaaaagcaagagccatggccaagaggacaaaggacaattttcatggttacgatgggattcctgttgaggaaaagacaaagaagaagcagctggtggagagccacctgaaccagggaactgaccaggagtatgatgtggaggaggaagacttcatagactacaatcaggctgagctggaccaggactatgaggaggagcaagagccacccaaggctgaaagcaaaccaaaggccccccttaaaagtgccccgtcacccatgaacttcactgacttactgaggctagctgagaagaagcagtttgaaccagtggagatcaaggtagtgaagaaagcagaagaccggcccctgactgcagaggaactgagggagcgagagtttcttgaacgaaagcataggaaaaagaaacctgaacccgatgccaaactacctccacccgtgttgaaaaaggcgccctctcataaagacatcatgggtacaaagcccagcagaggtgctggggacaggcagcttgcttccaaaggattgccctttcctcaagctgagaagaaattcagacccagcacggccagtgagaaacaagcagctttgtcttcacccaaatccctgccaggcgagaggaccaaggtagggtctggcagtagcacccagccctcacttcgggagggtcacaacaggcctgtcttcaatggggctggaaagccccgccccagcacctgttcaccaagtgtcccaaagactcctgctagcgggactcagaaatctgcttctgagcacaaagccaaaaaacctcttccttctcatccgagccattccaagcctggacccacggttctctcacacaacaaatctaagagtccaggtgtcagacaaccaggcagcaactctggctctgctcctgggcagcctaacccagggacagctcgacccacacttagttctggccctgtgcccaggcgccagaacggcagctccagctcaggacctgagcagtcagccggtgggatcagaaagctggccagcaattcacatctctccggacgaacactcaacggcacaaatggtcctggacgacctgcaagcagctcaagtggccctgggcgacccatcagtggatctgctggttctgggagacctgtgggcagttctggaggccccgggcagcctgtaaacaatccacatgaccttcgacgaccaatgaacagtcttagctctcctgggcgggcagtcagtggccctgggagatccataagtggttccattccagctggacgaactgtcaattcaggccctggaagaccagtgagcagcctaggccctggacgagcagttagtaacccaggcctccccacaaagcccagatgtactgttgtctcagaaacaatttcttcaaagaatattattagtcggtcaagtaatggacagataaatggaatgaaacctctcctctctggctacagatctgctcaaggtaaagttgacccttatcccaaagagtatatttatcactatgcctttggagaaagtacctagctttatataatcaggaaccctatgaccactttcacaagccctgtaatgtgctcattgttgatagtggaaaagaggcttatctaggacccgctgttcacaccccccacctctttctcacatgcagtattgtgatgtgtgagAGGGGGTGACTGGACGCAGCTGGTGGTTTGTAGGATAATGTTCTACATGAAGACTAAGCCAGATGAGAGGATGATTAAGAACTTAGAGCCTTTGCTGCTCTGTCATTCAGCAGGTGTTTATTGAACACCTCCCCTGTAGCAGCTGCTATTTTAGACTGTCCTGAGTTCTATTCCTTTTCATGGTGGTG
This mutation is a 2893 bp deletion beginning at Chromosome 7 position 46,997,270 bp and ending after 47,000,162 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46823 | AGT GGA AAA GAG GCT TAT CTA GGA C | Wild type Forward | A | |||
46824 | ACC AGC TGC GTC CAG TCA | Common | A | |||
46825 | CAG TTA CTT AAA AAC CAA AGT CC | Mutant Forward | A | |||
46826 | Fluorophore-1 | CCC CAC CTC TTT CTC ACA TG | Quencher-1 | WT Probe | ||
46827 | Fluorophore-2 | AGG ATT CAT GAC CTT GGG ATC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46823 | 0.40 uM |
46824 | 0.40 uM |
46825 | 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 |