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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:83434239-83434381 143bp CAGGGCAGTCTTTAGTAGCACAG CCATGACAGAACAATCTATGCAG
Mut = 164 bp
Wt = 143 bp
Fam = Mut
Hex = Wt
MUT Sequence (junction in uppercase):
taagtgcagcatttaacgtcagagtcttgattactgagcagctcagcagcatctgtccttatgcttctctctggaactagttgtcctttgctgatgtatgcatacacacttaatacttcCTtgttcccaggctagagggctctgctgcatagattgttctgtcatggcttctgcatcatcagttacactattcattctatttacaggaaagagtttgtgagtgcccagaatc
This mutation is a 3865 bp deletion beginning at Chromosome 8 position 83,434,286 bp and ending after 83,438,150 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50585 | CCA TGA CAG AAC AAT CTA TGC AG | Common | A | |||
50586 | CAG GGC AGT CTT TAG TAG CAC AG | Wild type Forward | A | |||
50587 | GTG CAG CAT TTA ACG TCA GAG T | Mutant Forward | A | |||
50588 | Fluorophore-1 | ACC CTT TAT TGA AGT TCT AGG GAA GG | Quencher-1 | WT Probe | ||
50589 | Fluorophore-2 | TGC ATA CAC ACT TAA TAC TTC CTT GTT CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50585 | 0.40 uM |
50586 | 0.40 uM |
50587 | 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 |