Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:82093116+82093212 97bp TGTGGTCACAACTAAGTCTCTATGG GGGACCCTTTTAAATCCTTAATCC
Mut = 102 bp
Wt = 97 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TCTCCTTTGTCTCTTTTTGAGACAGTCTCACTCTGGCCTGCATGTCCCAGCAATCCTCCTGTTTCAGTCTCTCAAATGCTGGGATTGTTTATAGGCACAAATCACGATAGCTGGCCTAATAATGCTGTTTGGAAACGTTATGAAATTTGTTCCTGGGAAGGCTGGTCTTGGTTGCCTTTAAGTTCGGGTCCCGGTGAGAGTATGGATCTGTGTCCCTGAccttacatagagacatgactcctgtctgatgccttctccttctctccctgcagaaaaccctgagcgagccgctctgtactttgtctctggagtgtgcattggcctgttcctgaccttggccgccttggtgatgaggatctcctgccacacggactgccggcgaggtccccggaggaggtgcttgcaggaccgcgagtgcagcgacagcagcgacagcgaggatggtagcgaggacacagcctctgacctctcggtgcgcagacaccgccgcttcgagaggactttgaataagaacgtgttcacctctgcagaggagctggagcgcgcacagcgcttggaggagcgcgaacgaatcatcagggagatctggatgaacgggcagcctgaggtgccgggcaccaggagcctgaaccgatactactagggctcagtgtgcaccccagaacctctgcagatcgccgggatgagagtgggttctgcaggcaaggacgatgtgcaagagctgaaaccagctctgttaatgtggctggagagtgcatagcgccagccagcctgtttccccaggagaatggggatttgctttagatccttgcctctggagaaatagacaacccaggcagtggcatctactctttcaggtcttgacagccacctgggcacaccagcccaatgctttcaggccttggaaaggtgctgaggaattcctctccaaccatgtgacagcgatgggacaagctgaaagtccttccgaagcgctcagaagtttgggcagacttatcctgtgctattattcctttataaaaggaaaaaaaaatctcatagctggagagctggggcatcagggtgaccgtaacccagaaaatgtaattttctaaaggactccgtgaagaggaaatggttttgattgagatagcatgatccatgctaattatttaaagctaatttttaaaacagaaaaaaaatcttgacaacctctgcattttaactgacacctcagggattaaaatcctatcttttagtctaggtcccacctcattcatgaagatgaatagaacttactgtattatgagatgtattggagacttaggatacatcaataacctcagaggataaagggttttattttttagtactctataaactatgtattgacatgtgtatttgaaaatgctgcataaaaatagaaaccgtgtgtcctttccttttggagagaaaagtctcatgacttcagagactcatgatatttaaataatattttgtttaaaaaaatagtttggatttccaagaatggaagccagaggtgagaattaaatccaaaattagaatggtatcgaaaataaacattccatgaaaatcttcttttgcttcgtgtctgtgtggtcacaactaagtctctatggttaacgaccttagttacaggcaagtatgacctctgcaggacTCCGGAAGGATTAAGGATTTAAAAGGGTCCCTGGAGTTAAGGGTTCGGGTTGTAGGTTGTAGGTTGTAGTTGAGTTATTGTTAAGCTGATAGAGGAACTGAAGGCTTAAACCTGAGGTAAGAGATCAGATAGAGACCCCACCCTCAGCCCCACCCCCTCTGTGGTGAGGAGAAATTTGTTGGACTCG
This mutation is a 1441 bp deletion beginning at Chromosome 6 position 82,091,741 bp and ending after 82,093,181 bp (GRCm38/mm10). There is a 1 bp (A) insertion at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50719 | GGG ACC CTT TTA AAT CCT TAA TCC | Common | A | |||
50720 | TGT GGT CAC AAC TAA GTC TCT ATG G | Wild type Forward | A | |||
50721 | TTC CTG GGA AGG CTG GTC T | Mutant Forward | A | |||
50722 | Fluorophore-1 | ACC TTA GTT ACA GGC AAG TAT GAC CTC T | Quencher-1 | WT Probe | ||
50723 | Fluorophore-2 | TGG ATC TGT GTC CCT GAA TCC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50719 | 0.40 uM |
50720 | 0.40 uM |
50721 | 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 |