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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:15976839-15976948 110bp ACAGTTTCCCTTCTGCCAAG GTGACCCTGACATCAAACTGG
Mut = 100 bp
Wt = 110 bp
Fam = Mut
Hex = Wt
MUT Sequence (junction in uppercase):
aaaagagcccagttcccaagtgcagtgcccagaacatccacccacacacacccatttcccatggctggccattCCatctccagtttgatgtcagggtcactgtttcttcagcttgtcaggtgtggtgggga
This mutation is a 3648 bp deletion beginning at Chromosome 7 position 15,976,865 bp and ending after 15,980,512 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
54693 | GTG ACC CTG ACA TCA AAC TGG | Common | A | |||
54694 | ACA GTT TCC CTT CTG CCA AG | Wild type Forward | A | |||
54695 | AAA AGA GCC CAG TTC CCA AG | Mutant Forward | A | |||
54696 | Fluorophore-1 | ATT GGC AGG CTG CCG AC | Quencher-1 | WT Probe | ||
54697 | Fluorophore-2 | CCA TGG CTG GCC ATT CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
54693 | 0.40 uM |
54694 | 0.40 uM |
54695 | 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 |