Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr18:36997754+36997904 151bp CATTTAGCCACTGGATGTCG CTTCTGGTCGATTTCCTAATCTC
Mut = 159 bp
Wt = 151 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
ACATTTAGCCACTGGATGTCGCTGCCGTCCATGAAATGTATTGCTTTTAAGAACAAAGATATAgttcgctgtttctcaggggataggaagtagctacgttcggagatctcggcgtttcggttaaaaaatgagattaggaaatcgaccagaagatattagaacttgtgtgcatctcagatggcacattcatggacttttaagacaagagaatgcttcagtggtgatctccaaatgtctgaggcatggagcttggcgcctgctactttggcttctgctccttgcaacatgggatgtagggagcggccagcttcactactctgtcccagaggaggccaaacacggcaccttcgtgggccgcatcgcgcaggacttggggctggagctggctgaactggtgccccgcctgttcagggtggtgtccaaggaccgcggggaccttctggaggtaaatcttcagaatggcattttgtttgtgaattctcggatcgaccgggaggagctgtgcgggcagaacgcggagtgtagcatccacctggaggtgatcgtggacaggccgctgcaggttttccacgtggaggtggaggtgagggacattaatgacaaccctccgatattctccgtagcggaacaaaagatcttggtagctgaatctagactgcttgattctcgatttccactagaaggcgcctctgatgcagatgttggagagaactctatgcttacttacaaactcagttcaaatgagttttttatccttgatattgtaaacaaaagaggcaaaggcaaatttccagtgcttgttcttcgaaaactgatagatcgagaagaaaatcctcagctaaagttgctactgacagcaaccgatggaggcaaacctgaatttacgggatcggtttctctgttgatccaggtgttggatgttaatgacaatgctcctgtgtttgacagatctgtttacgaagttaagatgtatgagaatcaagagaataaaaccttagtaatatggctgaatgctactgactcagatgaaggaataaacaaagaagtggagtattcatttagctctttagcctcatcgattataagacagaaatttctaataaacgaaaagactggagagataaaaataaacggcgctattgactttgaggagagtaacaattatgaaattcacgtggatgctacagataaaggatacccacctatggttgctcactgcaccgtactcgtggaaatcttggatgaaaatgataacgcaccggagatagtccttacttctttgtctttgccggtgaaagaagatgctcctttgggctctgtaatcgccctgatcagcgtgtcagacaaagactctggtgtcaatggacaggtgacctgctccttgactaaccatgtccccttcaagttggtgtccaccttcaagaattactattccctcgtgctggacagcgccctggaccgagagaccacagctgactataaggtggtggtgacagcccgcgacgggggctcgccctctctgtgggccacggccagcgtgtccgtggaggtggctgacgtgaacgacaatgctcctgcattcgcacatcccgaatacacggtgttcgtgaaggagaacaacccgcctggagtgcacatcttcacagtattggcggtggatgctgacgcacaggagaacgcgctggtgtcctactcgctggtggagcggagggtgggcgagcgcttgctgtcgagctatgtgtctgtgcacgcggagagcggcaaggtgttcgcgctgcagcctctggaccatgaggagctggagctgctgcagttccaggtgagcgcgcgggatgctggtgtgcctgccctgggcagcaatgtgactctgcaggtgtttgtgcaggatgagaatgacaatccgcccacgctgctcggacatcagtcaggtggacctgctggcgaatttagccagcttgtttcgcggtcagtgggtgcgggacacgtggtgtcaaaggtgcgcgcggtggatgcggactccggttacaacgcgtggctgtcttatgagctccacccaactgtgggcgctcgcagccctttccgtgtcggtctctacacgggtgagatcagcatgacccgagccctggatgaatctgatttaccgcgccagcgcctattggtgttggtgaaggaccacggcgaacctatgttgatcgccactgccaccgttttggtgtccctggtggagaacggccaggtgccaaaagcttcttcccaggggttgcccaactcctcccgcagggaggcgtcgctgatggatgtcaatgtatacctgatcattgccatttgcgcagtgtccagcctgttggtgctcacgctgctgctatacacagccctgcgctgctcagctgtgcccatgcaggctgggtgcgggcttgggaagcccactctggtgtgctcaagcgctgtggggacctggtcatactcacagcagagacagcagcgagtatgctcaggagagggtccgcccaaaaccgacctcatggcttttagccccagtcttacaccttgcccagtggccgaagtgggaatggaaagtcattctgttggaggagacgtgcccggaaaggtgggctattactttttactctcatcttgcttgatgaaatacattttctgtctcattttattttatgacaaatgtgtgtcccagatgtatttttttttgtctgagtaccactatgcacaaGTATTTTTAACAATGCGTTGGAATATAGTTTATTCCAGCAATTGGTTTTAAGATGTCCATCTA
This mutation is a 2665 bp deletion beginning at Chromosome 18 position 36,997,816 bp and ending after 37,000,480 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51580 | CAT TTA GCC ACT GGA TGT CG | Common | A | |||
51581 | CTT CTG GTC GAT TTC CTA ATC TC | Wild type Reverse | A | |||
51582 | ACA AGT TTT AGG CAG AGG TTT G | Mutant Reverse | A | |||
51583 | Fluorophore-1 | CGC TGT TTC TCA GGG GAT AGG | Quencher-1 | WT Probe | ||
51584 | Fluorophore-2 | CGT TGG AAT ATA GTT TAT TCC AGC AAT TG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51580 | 0.40 uM |
51581 | 0.40 uM |
51582 | 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 |