Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:28460010+28460117 108bp CCGTATGTACCAAGTGCAATACA GGACAGGATGTCAGGGTTAGA
Mut= 108 bp
Wt= 108 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TATGCTAGCTGCTTTGAAATCTGTTGTCCAGAGCCCATTCATTGGGGGATAAAAGATCCAGTTTGACTCTCAGCAGCACACCCCAGTTGACTATCTTCCTTCCCTCCAGGTCTCTCCCATTCCTGCCTCCCCCCTGCACCATGGCTccaggccccttctcctccgggctcttttcgccaccacctgctgctcttccctttctgctgttgctctgggcaggagcatctcgaggccagccctgccctggtcgctgcatctgtcagaacgtggcacctacgctgaccatgctgtgtgcgaagacgggcctgctctttgtgccacccgccatcgacaggcgtgtggtagagctgcggctcactgacaacttcattgcggctgtgcgtcgtcgagacttcgccaatatgaccagcctggtccacctcaccctgtctcgcaacaccattggccaggtagcagccggcgcctttgctgacctccgggctctccgggccctgcaccttgacagcaaccgcctggcagaggtgcgaggggatcagctccggggcttgggtaacctccgccacttgatcctcggcaacaatcagatccgtaaggtggagtcggcagcctttgacgccttcctgtccaccgtggaggacctggatctatcctacaacaacctggaggcactgccatgggaggcggtgggtcagatggtgaacttgaacaccctcacgctggaccacaacctcattgatcacattgcggagggcaccttcgtgcagctgcacaaactcgtgcgcttggacatgacctccaaccgtctacataaattgccccccgacggactgttcctgaggtcccagggcggtgggcccaagccacccaccccattgacggtcagcttcggtggcaacccgctgcactgcaactgtgaactgctctggcttcggcgcctgaccagggaggacgacttggagacgtgtgccacgcccgagcatctcactgaccgctatttctggtccatcccggaggaggaatttctatgtgagcccccgctcatcacacggcaagcagggggccgggccctggtggtagagggccaggctgtcagtctgcgctgccgggctgtgggtgaccccgagcccgtggtgcattgggtggcacctgatggacggctgctggggaactccagccggactcgggtccgtggtgacggaacgctggatgtgactatcaccaccctgagggacagcggtaccttcacttgcatagcctccaatgctgcgggggaagccacagcacctgtggaggtatgtgtggtacctctgccactgatggcgcccccacccgctgccccgccgcctctcactgaacctggttcttctgacatcgccacaccaggcagacctggtgccaacgactcaaccagtgagcgcaggcttgtggctgctgaactcacgtctagctctgtgctcatccgctggccggcccagaggccagtgcctggcatccgtatgtaccaagtgcaatacaacagctctgcagatgactccctagtctacAGGTGAGTGTGGTACTGCAGCTCCGGGACCTCCTCTCTAACCCTGACATCCTGTCCTCCTTTCCTCTCACCTGCCCATTCATCCCCCTTGACCTTGCTTAGTTCCAGCTCTGGATAGGCCTGTAGTGCCCCCCCCCCCAACGCAGTCTTTGT
This mutation is a 1398 bp deletion beginning at Chromosome 7 position 28,458,664 bp and ending after 28,460,061 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42878 | CCG TAT GTA CCA AGT GCA ATA CA | Wild type Forward | A | |||
42879 | GGA CAG GAT GTC AGG GTT AGA | Common | A | |||
42880 | CTT CCT TCC CTC CAG GTC TC | Mutant Forward | A | |||
42881 | Fluorophore-1 | CTC TGC AGA TGA CTC CCT AGT CTA C | Quencher-1 | WT Probe | ||
42882 | Fluorophore-2 | CTG CAC CAT GGC TAG GTG A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42878 | 0.40 uM |
42879 | 0.40 uM |
42880 | 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 |