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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mut = 82 bp
Wt = 95 bp
Fam = Mut
Hex = Wt
>chr18:37849443+37849537 95bp TGACAAGAATGCCCAAGTCA TACCCGTGTCAGAGTTGATG
MUT Sequence (junction in uppercase):
tccaagacctctcagggtgtgagcgagatcctcaggGCaggtgagttaatctctttaccttttctgtgtgcctgtgtgtaaagtgctctcatgttgccaggtttcttcttctattgtcttggga
This mutation is a 2435 bp deletion beginning at Chromosome 18 position 37,847,990 bp and ending after 37,850,424 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
57933 | CCA AGA CCT CTC AGG GTG TG | Mutant Forward | A | |||
57935 | CTT TAC ACA CAG GCA CAC AGA | Mutant Reverse | A | |||
57938 | Fluorophore-1 | CGA GAT CCT CAG GGC AGG T | Quencher-1 | MUT Probe | ||
58732 | TGA CAA GAA TGC CCA AGT CA | Wild type Forward | A | |||
58733 | TAC CCG TGT CAG AGT TGA TG | Wild type Reverse | A | |||
58734 | Fluorophore-2 | ACC GGT GTC GTC CTA CGT CT | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
57933 | 0.40 uM |
57935 | 0.40 uM |
58732 | 0.40 uM |
58733 | 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 |