Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:44460712+44460827 116bp CAGCCTTGGGTGCTTAATGA CATGCACCAATCGCTCACT
Mut = 100 bp
Wt = 116 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GCCAGCCTTGGGTGCTTAATGAAACCCTGTCTCCGAATACAAAgtaaaaagacagctgggaatgaaggataatggtagagcacctacctagaatcccccagtgagcgattggtgcatggctcagtggctattgaccatctagaatcctcagtcacagagtccctgtccagcatgtgagaggccctaggtcttgtccttaggcccacatactgatcgcagagtcgcgttcacctgctgtgaactggagcagcctgctgctcccacgcttgtcaggtgcactgacactggtctccccgcaggtgatccgcacggacacattcaagcacctccggcacctggagatcctgcagctgagcaagaacctggtgcgaaagatcgaggtgggagcgttcaatgggctgcccagtctcaacacactggaactcttcgacaaccggctgacaaccgtgcccacgcaggcctttgagtacctgtccaagctccgggagctgtggctgcgcaacaaccccatcgaaagcatcccatcctatgccttcaaccgtgtgccctcactgcgccgcctagacctgggcgagctgaagaggctggagtacatatctgaggcggccttcgaggggctggtgaacctgcgctacctcaacctgggcatgtgcaacctaaaggacatccccaacctcacagcacttgtgcgcctggaggagctggagctgtccgggaaccggctggacctaattcgccctggctccttccagggcctcaccagcctgcgcaagctgtggctgatgcatgcccaagtggccaccatcgagcgaaacgccttcgacgacctcaagtcgctggaggagctgaacctgtcccacaacaacctgatgtcgctgccgcacgaccttttcacgccccttcaccgcctggagcgtgtccacctgaaccacaacccctggcattgtaactgtgatgtgctgtggctcagctggtggctgaaggagacagtgcccagcaataccacatgctgcgcacgctgccacgcgcccgccggcctcaagggccgctacattggcgagctagaccagtcgcacttcacctgctatgccccggtcatcgtggagccacccacagacctcaatgtcaccgagggcatggcggctgagctcaagtgccgcacaggcacatccatgacgtcggtcaactggctgacacctaacggcacgctcatgacgcacggttcctatcgtgtgcgcatttcagtgctgcacgatggcacactcaacttcaccaatgtcaccgtgcaggacacgggccagtacacgtgcatggtgacaaactcggctggcaacactactgcctcggccacgcttaacgtgtcggccgtggaccccgtggctgcgggagggcccggtggcgggggcccagggggtgggggcggtgcagggggcgcgggcggatacacctacttcaccacggtgaccgtggagacgctggagacgcagccgggggaggaggcccagcagccccgaggtactgagaaggagcccccggggcccacgactgatggcgcgtggggcggcggccggcctgatgctgcagccccggcctcggcgtccaccacggcaccggccccgcgctcctcgcggcccacggagaaggctttcacggtgcccatcacggacgtgacggagaatgcgcttaaggacctagacgatgtcatgaagaccaccaaaatcatcatcggctgcttcgtggccatcaccttcatggccgccgtgatgctggtggccttctacaagctgcgcaagcagcaccagctgcacaagcaccacgggcccacgcgcactgtggagatcatcaacgtggaggacgagctgcccgccgcctccgctgtgtcggtggctgcggccgccgcggtggctggaggcgcgggcgtgggtggggacagccacctggcccttccggcgctggagcgagaccacctcaatcaccaccactacgttgcggccgccttcaaggcgcactacggcggcaacccgggaggcgggtgcggggccaaaggccccggcctcaactccatccacgaacctctgctcttcaagagcggctccaaggagaacgtgcaagagacacaaatctaaacccaggcgcacgggacgcgggacgcctgcccgagatgctcctgcttgccgagcaaccctggcggcgcctgcgggtcctgcaatgggaaaggacctggggaccaggagaggtggccccgccctccgccttgccgcgtcctctgccctcctgccctgcccgccaccttccaggggttgaggagcttcttcgaggggtacccttttctcctcaccccaacaaccctccttttatggctctttttttttcttttcttttcttttttttttttcttttgcagtttgacgcctgtccttccctcctactccttcaccctcaaaacacaagagacaggcaacttcaccagagcccagggggcaactccgacaatgcccccaacagcgagctccactcgcggccctggcaggcccaccccaccccacagactcttttgatactgaagggaggtttgcgtcaatcactacctgctctgtaattactttaaaaaaaaaaaaaaacacggaaaaagtaaaaaaaaatttttttgggggggggggcatgaaagtattgaagagaaacagggcacaaagctcctgagggtgggacgaggagaggataggcagccagcgatgctgtcctcccaggaggggagagtggatactccaaagttacagctctgggactctctatccccgagattccagagaggaacaggctcacccagcacatagcagcacggctccatctccccatGTGCTGGGGGTTCAGTAGCTGCCCTTCTGTGTCTCTGTCTCCATCCCCATGTCTAGCTGTTGTTCCTTTCTGACTCTGGTGCCTCCCCCATCACACACACAGCA
This mutation is a 2813 bp deletion beginning at Chromosome 7 position 44,460,753 bp and ending after 44,463,565 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
53247 | CAG CCT TGG GTG CTT AAT GA | Common | A | |||
53248 | CAT GCA CCA ATC GCT CAC T | Wild type Reverse | A | |||
53249 | CAG CTA GAC ATG GGG ATG GA | Mutant Reverse | A | |||
53250 | Fluorophore-1 | CAG CTG GGA ATG AAG GAT AAT G | Quencher-1 | WT Probe | ||
53251 | Fluorophore-2 | CAA AGT GCT GGG GGT TCA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
53247 | 0.40 uM |
53248 | 0.40 uM |
53249 | 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 |