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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:16873547-16873655 109bp GAAGAGCCTGGGAAGATGC TCTCAGCCGAATGCTGGA
Mut = 108 bp
Wt = 109 bp
Fam = Mut
Hex = Wt
MUT Sequence (junction in uppercase):
acttaagcctggtatccggctttccgagaggcgttaaggcccactgtgggccggacggaagtggaagagcctgggaagatgccacgGTtaggggaccccgcgaccggtgagaaggcgcctgtcgagagactgagacaatggtcccagaccgagagggtgctccaggaaggtcagcaaactt
This mutation is a 1274 bp deletion beginning at Chromosome 19 position 16,872,358 bp and ending after 16,873,631 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50701 | GAA GAG CCT GGG AAG ATG C | Common | A | |||
50702 | TCT CAG CCG AAT GCT GGA | Wild type Reverse | A | |||
50703 | ACC TTC CTG GAG CAC CCT CT | Mutant Reverse | A | |||
50704 | Fluorophore-1 | TCC TAC AGC GAC CAA AAG CC | Quencher-1 | WT Probe | ||
50705 | Fluorophore-2 | CGG TTA GGG GAC CCC G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50701 | 0.40 uM |
50702 | 0.40 uM |
50703 | 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 |