Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:92198517+92198632 116bp CCTCGAGCTACAAACCCAGA AGAGTGGCTGTTGGCAGGA
Mut = 120 bp
Wt = 116 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AGAAGCTATAGCTACAGGTTAGCAAGGGCCTGTTACCATTTAGTTTGCTTTTGTGAGCTATGATTCTTCATCTTCAAAAAAAAAATGCTACTTGTACAATGTATCTGCGGGTCAATTATTTAAGCCAAGTTATTCAgttatgtctccgggcaaccacaacattcttccatgttccttcttccctgactgagggcactgaatagtgaaaacggggaaaatgcgccaaaatgagagacatcaatcaaggtcaaaactgacacctaaatcaaattaaaaagaaaaagaaaagaaaaaagaatgaaagaaaaagaaaaagaaagaaaggaaggattatgtaaaatcaaaagcaacctaaactttaaatatccaacaagatatttatcttctcttgcagattcccagaaccaaaaccctagaagtctgcctgggtttcttcctcgagctacaaacccagatcaaaactctcaggaggcttcttccaatcccaggaaacaggcaaggcagcagccgggtgtctctcagccaagcagtttcctgccaacagccactctccctcctggtctagagtatttaagccaggtttgcatgacttcactttctactagagtgttttctttaaaagagcggtttcttgtttttcaaccaataatttaatggaatacaaaaatgcatgaaaatcatgaagttataaaagactgattttattgacttctattatttagaaagacttaatccactttattgccacctatccttatttacttgacagagtctcaagactaaacagtcTTGACTTTGTCATGCAACACGTTTATCTGCCTCTGCTCATGGCTCAAGGTGTTAAGATAGATATTCTCAAGGAATTATCAGACGAAACCACATACAGCTATTAATTAAGCATATTTATTGAACACTTGCTATATTCAAGGCTCGTAAGAAGCATAGCGCCTATCCCTTGTCACTCAAACATTTTGTAAGTTAATGATCAAACAAACAAACAAA
This mutation is a 953 bp deletion beginning at Chromosome 9 position 92,198,268 bp and ending after 92,198,820 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49628 | CCT CGA GCT ACA AAC CCA GA | Wild type Forward | A | |||
49629 | AGA GTG GCT GTT GGC AGG A | Wild type Reverse | A | |||
49630 | TTG TGA GCT ATG ATT CTT CAT CTT C | Mutant Forward | A | |||
49631 | AGA GGC AGA TAA ACG TGT TGC | Mutant Reverse | A | |||
49632 | Fluorophore-1 | CCA GGA AAC AGG CAA GGC A | Quencher-1 | WT Probe | ||
51386 | Fluorophore-2 | CTA CTT GTA CAA TGT ATC TGC GGG TCA A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49628 | 0.40 uM |
49629 | 0.40 uM |
49630 | 0.40 uM |
49631 | 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 |