Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:5241675+5241759 85bp TGACAGGCTGGATGAAATGA CCATTTTCCTGCCTTGAGATAG
Mut= 85 bp
Wt= 85 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TTTTAGATAGTCAAGTTTACAGGAGCTGTGAATTAGTGATGGGCTATTAGTGAGCTGTGTCATTATTTAATAAAAATGGCTTCTCTCACCTTATTTTTTATCCAGGTCCCTGACAGGCTGGATGAAATGAGATCCCCATGTAGCAATTGCcatggaaacctgtgactctcctcctatctcaaggcaggaaaatgggcagagcacatcaaagctatgtggaatgacacaacttgataatgaggtgccagagaaagttgcagggatagagcctgacagggaaaacagctcctcacatgacaacctgaaaacggatgagcgcaaaagtgaagtcttgctgggtttcagcattgagaatgcagctgccactcaggtcacctcagcaaaggaaataccctgcaacgaatgtgccacttcttttcccagtttacagaaatatatggaacaccactgccctaatgcccgccttcctgtcctgaaggatgacgagagtgagaccagcgagttagaggacagtgatgtggaaaacttaacaggggagatagtttaccagcctgatggatcagcgtatataattgaggactccaaagaaagtggacagaatgcacagactggggcaaacagcaaactcttttcaacagcaatgtttctggactccctggcatctgctggagagaagagtgatcagtcttctacggcccctgtgtcattctacccacagatcatcaacacttttcatattgcttcatccctcgggaaaccatttacagccgatccggctttcccaaatacctcagcattagcaggagttggtcctgtgttgcacagtttccgtgtctatgatctccgacacaagagagagaaagactatctaaccagtgatggctcagccaaaaactcctgtgtgtccaaagatgtccctaacaatgtggacttgtccaaattcgatggttgtgttagtgatgggaagaggaaacctgtcttaatgtgtttcttgtgcaaattgtcttttggctatatcaggtcatttgtaacccatgctgtacatgatcatcggatgactctcaatgatgaggagcagaggctcctcagtaataaatgcgtctccgccataatacaggggattggcaaagacaaagaacctcttataagctttctggaaccaaaaaaatccacttctgtttatcccaatttttctactacaaacctcataggacctgatccaaccttccgcggtttatggagcgcttttcatgttgaaaacggtgactctttgcaggctggctttgccttcttgaagggaagtgcaagcccttccagctcagcagaacagccgctggggatcacccacatgccaaaggctgaagtgaacctgggggggctgtctagtttagtagtgaacaccccaattacctccgtctccctcagccacttatcatctgagtctagcaagatgtcagagagcaaagaccaagagaacaactgtgaaaggccaaaagaaagtaccatcttacacccgaatgtggggtgccctgtcaaaagtgaacccactgagccgggagatgaggatgaagaggatgcatattccaatgaactcgatgacgaggaagtattaggtgaactcacagatagtattggtaataaagatttccctctcttaaaccaaagcatttctcctttatcatccagtgtgctaaaatttattgaaaaaggtacctcgtcctcctcggggactattgctgaagacactgagaagaaaaaacaggccgccgctgctggaagaagcaatggcaacgttactaatagctatagcattggtggcaaggactttgcagacggaagtatcagtagagatggcaccacagcagctcctagtgaaacgacccatggagacgaggacagttcaacaacacaccagcatggcttcaccccgagcacacctggtacaccaggtcctggaggagatggctccccaggcaatggcattgagtgtccaaaatgtgacacggttttggggtcttcaaggtctcttggtggtcacatgactatgatgcactcaaggaactcatgtaaaaccctgaaatgtcccaaatgtaactggcactacaaatatcagcagactctggaggcccatatgaaggagaagcacccggagccaggtggctcttgtgtttattgtaagactggacaacctcatcccaggcttgcccggggagagagttacacgtgtggctataaacccttccgttgtgaggtttgtaactactctaccactaccaaaggcaacctcagtattcatatgcagtcagacaagcacctgaacaatgttcagaatcttcaaaatggcaatggcgagcaggtgtttggccactctgctccaacccctaacactagcctcagtggctgtggaacaccctccccatccaaacccaaacagaaacccacctggcggtgtgaagtctgtgactatgagaccaacgtggcgaggaacctccggattcacatgaccagtgagaagcacatgcacaacatgatgcttctgcagcagaacatgaaacagattcagcacaatctgcacctgggcctcgccccggccgaggccgagctctaccagtactacctggctcagaacatcggcctgacagggatgaagctggaaaatcctgccgagactcagctcctgcttaaccctttccagttcgactccgccacagcagcggctctggcacCAGGACTTGGTTAGTACTTTCTGTTCTGCACTATTGTCCGTTTTGAATATTTTCCACTTTGATCTCTTGTAGGAGGGCACTGACAAACTGGGACTGTTCGCCAGAAAGGGCTTTCTCTTAAACTGCGTAGGCAAACTGCAAACAGGCACATTGCTTTAAAGCTAAGTTAAATCTAGAGAGTGAAGAGGTGAGAAATTAGTGACAGCA
This mutation is a 2576 bp deletion beginning at Chromosome 3 position 5,241,715 bp and ending after 5,244,290 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52460 | TGA CAG GCT GGA TGA AAT GA | Common | A | |||
52461 | CCA TTT TCC TGC CTT GAG ATA G | Wild type Reverse | A | |||
52462 | CAA AAC GGA CAA TAG TGC AGA | Mutant Reverse | A | |||
52463 | Fluorophore-1 | CAT GGA AAC CTG TGA CTC TCC T | Quencher-1 | WT Probe | ||
52464 | Fluorophore-2 | AGC AAT TGC CAG GAC TTG G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52460 | 0.40 uM |
52461 | 0.40 uM |
52462 | 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 |