Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:180714297+180714398 102bp AGTGGATGCAGGAATCTTGG TGAGGCAGACACCTTGCT
Mut = 92 bp
Wt = 102 bp
Fam = Mut
Hex = Wt
WT reverse primer anneals over the nucleotide sequence containing mouse genomic variation rs262999462.
Wt Sequence (deletions in lower case):
TCTTAGAAAGTGGATGCAGGAATCTTGGCCTGTGGTTTTACCAGACTCCTGTGgatgaggtggcgccagagcaagcacccctgtgaagatgcagcaaggtgtctgcctcacctgcaattgcctctgttgctctggcatcctgacctgtaatccctgccttgatgatgtcacagcacacaatgggctagcagtctgtgaccttcaggttgataggctaagccttttgtatagctgcaaagaaatgacagccttgactattaatcacaaaaatagtaaaaaccaagttcctaagaaactgtggtttatatgtaggatcatttaatgatggttgggctctttagaagtacaaagccttaattcctagttatctttcctatcagtgaagatagttttcttttcagagggctttaaggaagcagcagagaaatttcggatggagtctgggatcgaaccaagtgtggacctagaaacacttgatgagcgaatcaagattcgggagatgattctgaagggtcagatccaggaggccattgccttgatcaacagcctccacccagagctcctagacacaaaccggtatctttacttccaccttcaggtgagtttcagcagtctgatatggctgctagacagacaaattaagcttgtttgcagtggctcagaaagaaatcggccattgtgtgtaaggagacaggctttgagctagctgctgctttatgtactattgcaactagttactttttaaaggggggtgtgtactggtaataagaacacatgttccaccatgttcatagcagccttatttataatagccagaagctggaaagaacccagatgtccctcaacagaagaatggattcagaaaatgtggtacatttacacaatggagtactattcagctattaaaaacaatgaatttatgaaatttttggacaaatggatgtatctggaggatatcatccttagtgaggtaacccaatcacaaaagaagtcattagatatgcattcactgggggctggtgagatggctcagtgggtaagagcacccgactgctcttccgaaggtccagagttcaaatcccagcaaccacatggtggctcataaccatccgtaacgagatctgatgccctcttctggtatatctgaagacagctacagtgtacttacatataataataaataaatctttaaaaaaaaaaaaaaaaaaaagatatgcattcactgataagtggatatttgcccagaaacatagaacccccaagatacaatttgcaaaacacaagaaaatcaagaagagggaagaccaatgggccgatacttcattcctccttagaacagggaacaaaatacccatgaaatgagttacagagacaaagtttggagctaagaagaaaggatggactatccagagactgccccacccagggatccatcccataatcagccaccaaacccagacactattgcatatgccagcaagattttgctgaagggaccctgttatagctgtatgaggctatgccagtgcttggcaaatacagaagtggatgcccacagtcatctataagatggaacacggggcccccaatggagaagctagagaaagcacccaaagagctaaaaggggtctgcaaccctataggtggaacaatatgaactaaccagtacccccagagcttgtatctctagctgcatatgtagcagaagatggcctagtcggccatcactgggaagagaggccccttggtattgcaaactttatatgccccagtacaggggaatgccagggccaagaagcaggagtgggtgggtaggggggcagggtgggggggagggtatagggaactttcaggatagcatttgaaatgtatataaagaaaatatctaacaatcaatcaataaataaatggataataaaaactatacttttatcaaggaaaaaattaaaggggtgtatgtgtctgtgtgtgtgtgtgtgagagagagagagagagagagagagagtatgaatgaatagtgtacatatggaaataagaggcattccttgggagtccattctttctgcccttacatgggctctgggcatacttgcacaacgagcactttcactgtggcagtctcaccaatctctccttttgcttttaaaatggaaatgtgccaggcgtgatggcgcacacctttaatcccagcactcgggaggcagaggcaggtggatttctgagttcgaggtcagccagggctacacagagaaaccctgtctcaggggaaaaaaaaatggaaatgcatcttggagatgctaagaaagtttcttttaagatgcctttcttattttcttttagggtgatggggtggagcccaggcattgcctagatgctaaacaagtactttaacactattagttagacactcaatccacacataagatatacttttgattggagccaagtgttgatggttattcatctaagtgtatgccagccaaacttggttgtctttgctgttgaccatactgggttccacctactgggtgtacaggctggctaatcttgtttgtctttgttttgactagcaacaacacttgattgagcttatccgtcagcgtgagacagaagcagcattggagtttgcccagacacaactggcagagcagggtgaggaaagccgagaatgcctcacagagatggagcgcacactggccttgctggccttcgatagccctgaggagtcgccttttggagacctccttcacatgatgcagaggcaaaaggtaaagggccagtaaaggaagaacagagtgcttagacactcagaggacttgtgaaaagatggtactgtgctgcgcttgtgttggggtgaagcacatggctctgggccaggatactccagtagaacactggcagctccactgatctgtccccttttaccttgtgagagGAAGAGCAGCGCCATTGTGACTGTTAGTGTCTAAAACCCT
This mutation is a 2882 bp deletion beginning at Chromosome 2 position 180,714,342 bp and ending after 180,717,223 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
53056 | AGT GGA TGC AGG AAT CTT GG | Common | A | |||
53057 | TGA GGC AGA CAC CTT GCT | Wild type Reverse | A | |||
53058 | GTG GCA AAG GGT TTT AGA CA | Mutant Reverse | A | |||
53059 | Fluorophore-1 | CCA GAG CAA GCA CCC CTG | Quencher-1 | WT Probe | ||
53060 | Fluorophore-2 | CCT GTG GAA GAG CAG CGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
53056 | 0.40 uM |
53057 | 0.40 uM |
53058 | 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 |