Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:47710961-47711122 162bp CTCAAGAAGAAGGTGGTGGTG GGATTCAGTGCCTACTTGGA
Mut= 174 bp
Wt= 162 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GCCTATCCTGGAACTTGCTCTATAGACCAGGCTGACCTCAACCTCAGGGGTCATGCCTGCCTCTGCTTCCTGAATGCTGGGATGAAAGGCACTTTGCTGCCACCCCACTCTCTGTGATTTTTAAATGAATGTTTTGTTGATTGTCATGTCTTTAGGACTCTTAGTAAAATACCTGTGTTTTTCTTCTTTCTAGAAGGGGTAGAGGAGCCATCcaaaaaacgagccgtagaaggaaaaaacaattcttcagttgagcgagatcatgggaaaaaatctgccaaaacggatcgttcagcagctcagcaagaaaacagttcgccgtccagggggtcgtccaccaaatccgagagtggtgggacctcagctcggagcagctccagtggctcccatcaggatagcgccacaagtgaaggagacagatctgtgtgcagccagagcagcagcaattcctctcaggtaaccgcagggtctggaaaggctttggagtcagaagctccacataaacgtggttcagcgtcatttgtttcttccctgttgaagtccagtatgaatagtcatatgacccagtccactgataacagacaacagagtgggtcacctaaaaagggtgctctggaaggttcttcaggctcagcctctcagagcagctcagagattgaggtgcccttgttgggctcctctggaagtgcagaagtagagttgccattactgtcttgtaagtctagttcagagacagcttccagtgggttaacatctaaatctagttcagaggcaaacatttcatcatcagtttcaaaaaacagttcatcgtcaggcagctctttactaaccccccagagcagttccacaaatccatcgctgctgacatccaaaagcacggctcaggtagctgcttcactgctagcgaccaaaagtggcgcttccttaggaagcgtgtctcagctagctgccaagagtggttctcagagtagcacttcacagttgccttcgaaaagtacctcccaggccagtgagagttctgtcaagtttgcttgtcggaagttaaccaatgaagacataaaacagaagcaacctttcttcaatagactgtataagacggtggcgtggaagttagtagcagttggtggcttcagccccactgtgaatcacggagagctgctgaacgcggctattgaggctctgaaagcaacactggatgtgttttttgtcccactgaaagaactggcagatctgcctcaaaataagagctctcaagaaagtattgtctgtgaattgaggtgtaagtcggtatatttgggcactggctgtggaaaaagcaaagaaaacgcaaaagccgtcgcatcgagagaagcactgaagttatttctcaagaagaaggtggtggtgaagatctgtaagaggaagtaccgaggcagcgagatcgaggatctagtactcctggatgaagaagcaagacccgtaaacttacctccagCACTGAAACATCCTCAAGAATTACTGTAACTTCTCCAAGTAGGCACTGAATCCAGTATCTGGTGTTTGAAGAGAAAAACTGGCTTTTATATAAGACACTGGCTTTCACAGATTTTATATTGCTTTTTTCCAGTTTTGCAGAAAATTTAAATTCTCGTTCTCTTCACTAAAGTAGAGCTGTAAATAATTTATGACAGTGCATATTAAAGGGTGT
This mutation is a 1262 bp deletion beginning at Chromosome 8 position 47,711,014 bp and ending after 47,712,275 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52805 | CTC AAG AAG AAG GTG GTG GTG | Wild type Forward | A | |||
52806 | GGA TTC AGT GCC TAC TTG GA | Common | A | |||
52807 | CTT TGC TGC CAC CCC ACT CT | Mutant Forward | A | |||
52808 | Fluorophore-1 | AGA GGA AGT ACC GAG GCA GCG | Quencher-1 | WT Probe | ||
52809 | Fluorophore-2 | AAG GGG TAG AGG AGC CAT CCA C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52805 | 0.40 uM |
52806 | 0.40 uM |
52807 | 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 |