Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 80 bp
Wild Type = 110 bp
>chr17:25268957-25269066 110bp GATAAGGAGTGGCCTGTGGT TTTGTAGCCAGCAACCTTCA
Wt Sequence (deleted region in lower case)
TCTCAGGGCTTTGTAGCTGTCCCAACAAAGAACCCTGATGGCACAATGAACCTGATGAACTGGGAGTGCGCTATCCCTGGAAAGAAGGGGGTAAGGGTGACTGCTGGCTTTGCACCTACTAACTTAACAGATCTGAAGGAGCAGAACTACTAGAAGGGACCTAAGTTTGCATATTTGTGACCACAGCTAGCCTGCCAGGTCACAGCTCTCCCCTGGATAAGGAGTGGCCTGTGGTGGTAAGGAGGCTTGgaccttggcaaatcactggatcagtgaacagcccggggtcaggggtaaagagcaggtgaaggttgctggctacaaagcagtaagccccaatggtgaggtgcccatggagaacccaggccgtgtttctttacactcatgggcctgagtgcagccggttgtctgcccttgtccagcacatgtaagagggatggtctcctataacttcaggcattcaccaaacccagacctctaggtcagcttagtgtctgagcaaaaacatagtttgggttcatctatacaaggctcagttgcagagtatgcagtaatggtggtgtagggcagttagagaagaagcccaaaggacccaggtttcaggaaacttctcctgggcttaccttggcaagtgagccactggtctaaccttggttccctccccacagactccatgggaaggaggcttgttcaagctacggatgcttttcaaagatgactatccgtcctcaccaccaaaatgtaagttgtgcaaagagcctgcagtccacttcccagcactaccaccaccctttgggagccatgtctgtgagtgcttggcctagtaggcaggtgccagtgctgggatagtcggtcctggtgagttgtcttggatgacaagtcccagggtagatgcgcatgtaattagtgtggtctctaatcccagcctattaggatgtaccacacatggcagagggagagttgtgtcaggctgaagcttctgggtgcttttgtgcccacaggagaactcgagtctgtaagttcccatagattacgtgagtggttagcatatatcagctgagttccattcagagcactaaaggagagaggagactcttactcatccccatctgctgtgttctgtgggatgctcatggccttcagaccatggggtcagaagagaaggctggactgatctctgttcagctcagcagaacttgcttgGGGGTACAGTGCCCACCACCACCATTCTGCCCAGGATAAGTAAGGCCAGGTCTGAGCTGGTTTATAAATAGATGTTACCTGATGCAGAGTTCCCCAGGAGTACTAAAACATAGTTTATTGGGAACCACTAGATGGAGTCTTGCCTGTTCTGTTTGGGATTTCCTTGTCTGCCTGAGTTCTTTTGTTAGTTTTTTCTTTTAAGGGGTATGTAGCTAGGATTGAGGTAGACAGAGTTGTTGGTCTCTTTCCTTTCGTGTGGTGCTGGA
954 bp deletion beginning at Chromosome 17 negative strand position 25,269,032 bp and ending after 25,268,079 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37953 | GAT AAG GAG TGG CCT GTG GT | Common | A | |||
37954 | GCC TTA CTT ATC CTG GGC AGA | Mutant Reverse | A | |||
37955 | TTT GTA GCC AGC AAC CTT CA | Wild type Reverse | A | |||
37956 | Fluorophore-1 | CAG TGC CCA CCA CCA CC | Quencher-1 | MUT Probe | ||
37957 | Fluorophore-2 | TGA ACA GCC CGG GGT C | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37953 | 0.40 uM |
37954 | 0.40 uM |
37955 | 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 |