Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:98716402+98716500 99bp TGAACAGCACCTCAAAGCTC TACATGTCAGCATGGTGCTCT
Mut = 95 bp
Wt = 99 bp
Fam = Mut
Hex = Wt
WT Sequence (deletions in lower case):
TTTGTGGATTTCATTTCTATCCCTGTAACTGTCTGTATCTGTGGCCTTGGGCAAGTTATTTACAGTCCTTATTTGTCAGAGGGATTGATACCATCTTCCTGAAGAAAgtaacgtgatccccgtaaagtgctggccacagtgcctgtaggaatggttgttctcctcaccttcctttttctcatggcatggattttgtttattaaaatctgtctgcagcatttgtccctttctaacttatttttgtgtcctactttgttaatgttctattagtggtgaatgaatgcatttgtggattttaaatatttagccaatcaaaagtaatttgttttgtcgtttgtggttcttcatgagtctgtttataaaagtaactttttgttttccagttgaggagacttctgaacaagagcaggaaactaacaccaatgctcagaaccccagtgcagaagcggtgaaccagcaggatgcaaatgtgctgcctttaacccttgaagagaaggagaacaaagagtacctcaagtctttatttgagattctggttctcatgggaaagcagaacatccccttggatgggcacgaggctgacgaggtccccgagggcctctttgccccagacaacttccaggcgctcctggagtgccgcatcaactctggcgaggaggtcctaaggaagcgctttgaggccacagcggtcaacacattattctgctctaaaactcagcagaggcacatgctggagatctgtgagagctgcattcgggaggagactctcagggaagtgagggactcacacttcttttccattatcacagacgatgtggtggacatagcaggagaagagcacctgcccgtgctggtgaggtttgtcgatgacgcccataacctgagagaggagtttgtgggatttctgccgtatgaagctgatgcggagattttggctgtgaaatttcacactacaataactgagaaatggggattgaatatggagtattgtcgtggtcaggcttacattgtgtccagtggattttcttccaaaatgaaagttgttgcttcaagacttttagaaaaatatccccaggctgtctataccctctgttcttcctgtgccttaaatgcatggttggcaaagtctgtccctgtgatcggggtgtccgtggctttaggaaccattgaggaggtctgttcttttttccatcgatcaccacagctgcttttagaacttgacagtgtaatttctgttctttttcagaatagtgaagaacgggctaaagaactgaaggaaatttgtcattcccagtggacaggcaggcatgatgcctttgaaatcttagtggatctcctacaagcacttgtcttatgtttagatggtataataaatagtgacacaaatgttaggtggaataactatatagctgggcgagcatttgtcctctgtagtgcagtgacagactttgatttcattgttaccattgttgttcttaaaaatgtgttatcttttacaagagcctttgggaagaacctccagggacagacctctgacgtcttctttgcagccagcagcctgactgcagtactgcattcacttaatgaggtcatggaaaatattgaagtttatcatgagttttggtttgaggaagccacaaatttagcaaccaaacttgacattcaaatgaaactcccagggaagttccggagagcccagcaaggtaacctggaatctcagctaacctctgagagttattataaagacactctcagtgttccaacagtagagcacattattcaggaacttaaagatattttctctgaacagcacctcaaagctctgaaatgcctatctctggtaccctcagtgatggggcagctcaaatttaacacatcagaagagcaccatgctgacatgtacagaagtgacctacccaaccctgacacgctctctgccgagcttcactgttggagaatcaagtggaaacacagagggaaagatatcgagctcccgtccaccatttatgaagccctccatcttcctgacatcaagtttttccctaatgtatatgctttgctgaaggtcctgtgtattctccctgtgatgaaagttgagaatgagcgctatgaaaatggacggaagcgtctcaaagcatacctgcggaacactctgacagaccagaggtcaagcaatttggctttgcttaacataaattttgatataaaacatgatttagatttaatggtggacacatacatcaaactctatacaaataagtcagagcttcctacaattaattcagaaaccattgaaaatacctagaagattttaaaaatggctgttatcttattttgctgtttggaagaaaagctgtaaggtatagtaggccagataatgacttgctgatagacctccagtttaacatactattagctattggtaatccacgtgtttaaatggcccctgtttgaacactcatgctttcaatacctgtctgttcttccagaagttggcattaggagtgccaaaatacctctgctggtggcactctgaaaatgttttacttaaaagtcgttttaggggctggagaaatggctcagcggttaagagcactgactgcctttccagaggtcctgagttcaagtcccagtgaccacatggtggctcacaaccatctgtaatgggatccgatgccctcttctggtgtgtctgaagagagcaacagtgtactcatatacgtaaaataaataaatctcttaaaaagtcattttagatatgacattatcactgtcgatccagttgtcagttattaagttatcaggtctttggaaaatgaattttgtgagagaaatacattttataatgtgtcatgatgaaaaacaccattgactttgacttggagtttcaggtgtttccgagctgtggtagcagcacttgggaggcagaggcatgaggatgacaggctttaggcctggaccacatagtgagcctgcttcaaagaacgagtgagggctgactggggtggagcctagtgtggagctcttgcctcagtactcaaaagcaaaaagaacagcaaaagtcacaggaagtaacgagcagccttctgtgtagatcctgtcagggcagaagaacctcaaaggaacaagttacagtagcagagccagccctgctgctcctcacttgattgctgctgtttgcatttctttgctgagcccacattttcataggcttgtttgctattcacttgtgcttcatggagaagaaagacccagaggccatccctataggcagctgacttgtctgttgtctccttacatgactgtgtctgctgccttggctcggttataagcaaaacctacagaaataaatgttttgtggtttatctaaaaataatacagaaaatattgctgttatttttggtgaataaaatcaattttgtatagtttatttcaacctaaataaaatgtgaattttgtttaaatttcaggtgaggtgtttttggtgggtcagaatacatccttgtatattttcttaaaATGGTTATTTCATAATCAGCCTCATGCCTTTTACGATTCAAGATGAAGTATTTTCTAGCTTTTCTCTT
This mutation is a 3412 bp deletion beginning at Chromosome 7 position 98,714,706 bp and ending after 98,718,117 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
54669 | TGA ACA GCA CCT CAA AGC TC | Wild type Forward | A | |||
54670 | TAC ATG TCA GCA TGG TGC TCT | Wild type Reverse | A | |||
54671 | GGC CTT GGG CAA GTT ATT TAC | Mutant Forward | A | |||
54672 | AAG GCA TGA GGC TGA TTA TGA | Mutant Reverse | A | |||
54674 | Fluorophore-1 | ATG CCT ATC TCT GGT ACC CTC AG | Quencher-1 | WT Probe | ||
54675 | Fluorophore-2 | CAG AGG GAT TGA TAC CAT CTT CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
54669 | 0.40 uM |
54670 | 0.40 uM |
54671 | 0.40 uM |
54672 | 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 |