Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:15719906+15720024 119bp AACATCATGGGGATTTCTTCA CTGTCCTCAGGCTGCACAC
Mutant= 100 bp
Wild Type = 119 bp
Wt Sequence (deletions in lower case):
TGTCAGGGATCAGAACTCAGGTCCTTAAGCTGAGGGTCAAATGCTTTATCCAGCAAGGCAACTCTCTAGCCTGATACTTCCTAATATTTAAACATTCACAAGTTGTAGCCACACATCAGTTTTACCAGcagtggtgcaggaccatatttttttttctgttttatagatatggacgtgatgaggcccttaataaacgagcagaatttcgatgggtcgtccgacgaggagcaggaacagacgcttgtgcccatacagaagcactaccagctcgatgggcagcacgggatttcgtgagtgtcacggtcacgggtccttagctctgttaccgcagcctgcatttctcagtctcccataggtctgagtgttttatgactaagaagattaaggagtcagactcctcaacacttatgtctatgagggagggacattaggctgactgaacatcatggggatttcttcagtagtgatagataccaaatgctgtgtccctcagAGGAAGATCTCAGAAGAGGGAGGGAGCTCGAGGGAGTGGGGAATGGGTGTGCAGCCTGAGGACAGGCTACAGGTGGAATGTTAAAAGCCTCT
A 365 bp deletion beginning at Chromosome 9 position 15,719,595 bp and ending after 15,719,959 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49682 | AAC ATC ATG GGG ATT TCT TCA | Wild type Forward | A | |||
49683 | CTG TCC TCA GGC TGC ACA C | Common | A | |||
49685 | Fluorophore-1 | TAG ATA CCA AAT GCT GTG TCC CT | Quencher-1 | WT Probe | ||
51000 | ATC CAG CAA GGC AAC TCT CT | Mutant Forward | A | |||
51001 | Fluorophore-2 | CAT TCA CAA GTT GTA GCC ACA CAT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49682 | 0.40 uM |
49683 | 0.40 uM |
51000 | 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 |