Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:13559852+13559973 122bp CCCAAGGTACACAAGAATTTCA GTTTCAGGATGCCCTTAGCA
Mut= 114 bp
Wt= 122 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
TTCTGGTAAACTCAATAAGGCTTTGCTTTAGAGTGAGGTCAGTTGAAGATATATTTGAAACTAGAGCATAAGCTTATGAAATTTGAAATCTTCCATTAAAATATTCAGAAATGGTAGGTCTTTTTTTTGAGACTTATCTCCTCTGTAATAATTTAATGAAAAACAAATTTTCCCAAGGTACACAAGAATTTCACTAAAAATATAGCAAGGTTTGTGTAGTACAGTGGATTAGACATCCCTgttggtgagtttatgatatttgcactggggaactgctaagggcatcctgaaacccatgttctaatacagtcagaaatggtaggaatgccttttgaattgacattttgcatattttaaagacaccagctgtacgaaacaaacaatgtcaatacaactctctctctctcccccccccctctctctctctctcaaggctactcggcatgctgaaatggtggccattgatcaggtcctagactggtgtcatcagcatggacagagcccttccactgtattcgagcacaccgtgttgtacgtcactgtggagccgtgcatcatgtgtgctgccgccctgcgccttatgagtatcctttggcttctagacttcctgcctttaccttgcctgtgagcacaacatggccagattcacgacccattgttctttctttttagtgcttgtttttcttttcaacgttgagaatggaagccaggctcttGTACTTGCTGCATCTGCTCTGTCCCTGAGCCATAGCCGTCCTTAGCTTTCAGTTCCTTAATGTGTGGAAACAGCGTTATGAAATACACTCTACAATTTAGCCAGAAACAGGAAAACCAGTTTCTACACAGCAGAGTGACCTGTTCTTTCTCTGTATCGACAGCCATGAAAGAATTGAATACTTAAGCAAAGGGAAGAAAAGCTCTTTTGTCAGGATAAATCCAAATTAAGGTGGAGCACAGG
This mutation is a 476 bp deletion beginning at Chromosome 10 position 13,435,665 bp and ending after 13,436,140 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
64690 | CCC AAG GTA CAC AAG AAT TTC A | Common | A | |||
64691 | GTT TCA GGA TGC CCT TAG CA | Wild type Reverse | A | |||
64692 | CTA AGG ACG GCT ATG GCT CA | Mutant Reverse | A | |||
64693 | Fluorophore-1 | TGG TGA GTT TAT GAT ATT TGC ACT G | Quencher-1 | WT Probe | ||
64694 | Fluorophore-2 | AGA CAT CCC TGT ACT TGC TGC ATC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
64690 | 0.40 uM |
64691 | 0.40 uM |
64692 | 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 |