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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mut = 111 bp
Wt = 106 bp
Fam = Mut
Hex = Wt
>chr12:78961590+78961695 106bp ACTGCAGGAGTTAGGGAGCA AACCAGGATGGCTGGTGGT
MUT Sequence (junction in uppercase):
acctccagcctgggactgcaggagttagggagcattggagtggacacaggtgACtaccttctgtatcacctcaactccctgttagcactggctttcttctgagaaactctacgcaaatactatgtgaaaatgtctactgaa
This mutation is a 3696 bp deletion beginning at Chromosome 12 position 79,008,403 bp and ending after 79,012,098 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
57776 | ACT GCA GGA GTT AGG GAG CA | Common | A | |||
57777 | AAC CAG GAT GGC TGG TGG T | Wild type Reverse | A | |||
57779 | Fluorophore-1 | TGG CAG CGC CAC ACC | Quencher-1 | WT Probe | ||
58735 | ACA TAG TAT TTG CGT AGA GTT TCT CAG | Mutant Reverse | A | |||
58736 | Fluorophore-2 | CTC AAC TCC CTG TTA GCA CTG GC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
57776 | 0.40 uM |
57777 | 0.40 uM |
58735 | 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 |