Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:119578941+119579060 120bp GACCAATGGAGAAAGGAGGA CTGGGATTGCTGCTGTGA
Mut= 103 bp
Wt= 120 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CCAGTCTACCCGAATCAGTAGTCTCCAAGTTCAATGAGAGAGAGAGTTCATAGTTCAAAAGCTATGGCAGATATGATAGAGAAGACAGCTGGTGTCACACCTCTGGCCTCTGTGTGACCTTGCATACCCAGGAACACACACAAACAGACCAATGGAGAAAGGAGGACGAGGAGGACccgatgtgaggcttcagattgaaaactttagacagaatggatggagaaaagagcctgagcgggtgccatggctcacagcagcaatcccagcggtggcgcaggcaggagagccacaagcttgaggccagcctgggttacctactgagttcccagctgaccctgtctccaaactaaatggagccaaatacaaacccttaactattttttttcacagacgacaagctgaagtgagcgtcttgtgttttgcttttcaacaggatgacgaaacagttccttcaaaagctctgcaagcagcacaagctgtacgtcacccccgccctgaacgacaccctgtatttgcactttaagggtgagtcccaggcgagtctatagccagcgtcctgcagaaaggggggagctggagagcatggcttgcagaggttgtcttcgcgctcagacgctatgcaagtggagctcggtcttgactgagttcctaaagggcCTTCTGAGTACAGTGGAGCTGGCCCGCCTTCTTTGGTTGCTTGGTTTTTTTCTTCCTTGCAAAATAGGGATGGAACCCAGAACCTCAAACATGCTAAGCAGGCACACTGCCACCAGATACAACTTCAGTTAGCCCGCCTTCCCCATTTTATTTTGAAACAAGGCTCACCAAATTGCCCTGGC
This mutation is a 481 bp deletion beginning at Chromosome 8 position 119,578,971 bp and ending after 119,579,451 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47018 | GAC CAA TGG AGA AAG GAG GA | Common | A | |||
47019 | CTG GGA TTG CTG CTG TGA | Wild type Reverse | A | |||
47020 | CCA TCC CTA TTT TGC AAG GA | Mutant Reverse | A | |||
47021 | Fluorophore-1 | ATG GAT GGA GAA AAG AGC CTG | Quencher-1 | WT Probe | ||
47022 | Fluorophore-2 | AGT ACA GTG GAG CTG GCC C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47018 | 0.40 uM |
47019 | 0.40 uM |
47020 | 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 |