Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:38225815+38225920 106bp GGCCAGGTGTGGAAAGTCT TGCACTTCACATGGCTTTTG
Mutant= 106 bp
Wild Type = 106 bp
Wt Sequence (deletions in lower case):
CCAGGTGTGGAAAGTCTTTGAGAGCCACTAGcatgctgtcagcctagtgaccagccctgggtccctctgcaggggtgatgactacaaaagccatgtgaagtgcatcagtgaaggtcagaagtacggaggcaaaggctatgaagccaagacacacaaaggtgatgcaaaacagcaggcatggattcaggtaagggtccctcccatcctactgctcatccctacctgcttcctcctgggggctcagcagatgggagtccctgagacagcacgtcttagagtgcagcagaagtgtcagaacatcaggaaagataaaattgtgctgcatttatatctggttcgtgtttgctctcaagatatctgagggttcctgctaacttccttttggctgtaaacaaaatcaaagtgtacaggtacacttggcagtggtagaggtcctatagcttagccgtgtcagcctgtcacagctgaacaaagtaaacatttttacaaatttaaggaattttccagtgaaaatatgtacaaaagcagcaagccctggtttgcagcaagagaagatgggtgggcagatgtcacctttgtacagactgcattctcctggaaatcagattagttgttcccccactgaatggctcagagtgagtgtgaacttggggtggtggctctctctgaaggtgtatactgctggcctgaccctcatgagggacttgaaggaggagctgcctggttgagtaccacttgggaagctgctgaaacctttaggatgtgtgtagtgctggcgagatggttgagctcggaccctttcccactgagctgtcctgctgacctttacttaatactgtagtctgccatgtgtaagaacccattatcaggtcagaaggcgcctcctctgccaagggtttttgtgttgttttggtttggttttgtttgtttgtttattggtttttcgagacagggtttctctgtgtagccctggctgtcctagaactcactttgtagaccaggctggccttgaactcagaaatccacctgcctctgcctcctgaatactgggattaaaggcgtgtgccaccacgcccggcttgcaaaggtttgtatagtttcactttttaaaatgtagatccttgattcattttgaggtgttttttgtataacataaggtaagcatctaatttccctgtggggatccactatttctgtcatcatttgttgaaaaggggatctgttctcccatagactcttcttgacatccttgttgaaaagccccttgaccatatcctcaggggtttattcctgcattttctgttcttttccagtggtctttgtggcagtaccacactctttagtttattgtagctttgtagtaagttttgaaatttaaaaaaaaaaacatttttcaaaattgttttgttgtttggagtccatcagggttccatgaattagagcacacatatgctagtctttaccagggctttcaggttaacatctaaatgttgtcagtgcatgaatgccgtttttattcttgtaactaactgtccagtgcccagttccttcaccgagttggttatggttctttatgttgtgctgcagatggaattctcctaatgttcacgtctgaattggtttttttgttgtttggttggttggttggtttttgtttttgttttttggtgtataaaaaagcagcttgttctttaagaggtgagtgtgtgtcctgcagctctgctgaatgcgttgttaattctaactggctgtgtcctgtagcactttctacatgcaagaccacattccctcaaatactgcctgtacacgtgtgacctgattttgttctttctgttaggggggctttggcttctttttcttgcttaaccactttggccaggactccagtgctgtgctgaataggagaatgtttgtcactatgcatagatgtaaatatagagtcttagtttaaaatgtgagagccctttggccatgccaaggacttgagtttggcttcatgtaattatgtcacgtgtttattttcttctatcttaaatagaaaattaatgagttaataaagaaacccaacgtcagccccaaggtgcgagaacttttgcagcaaatcagtgcttttgataatgttcccagaaaaaaggcgaagtttcaggtatggatttaaatgactttcctaaaccatggtgtgacccatctgtggcctgcagaggaacagtggctctttgtcttgtgtccacagaactggatgaaaaacagcctgaaagtgcacagcgactccgttctagagcaggtgtgggatatcttctccgaagcatccagcagtgtgagtcaactccactttgccactgcaatatggcagagaactgggaataaggaaatggggtgccatgtgtggggatcaagatggcatttccagggaAGCTCTGTGCTCTCTCCCTGATGGGCAGGAATGATCTGGGAGCGCCTGTGGTGCAGAGACAGC
A 2382 bp deletion beginning at Chromosome 5 position 38,225,848 bp and ending after 38,228,229 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47942 | TGC ACT TCA CAT GGC TTT TG | Wild type Reverse | A | |||
49612 | GGC CAG GTG TGG AAA GTC T | Common | A | |||
49613 | AGG GTG AAT GGC TGT CTC TG | Mutant Reverse | A | |||
49614 | Fluorophore-1 | TGC TGT CAG CCT AGT GAC CA | Quencher-1 | WT Probe | ||
49615 | Fluorophore-2 | AGA GCC ACT AGA GCT CTG TGC TC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47942 | 0.40 uM |
49612 | 0.40 uM |
49613 | 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 |