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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:119909518-119909636 119bp CCCTTTCTTTCTTGCAGTGG TCACAAGTCTCCTCACCTTGTC
Mut = 115 bp
Wt = 119 bp
Fam = Mut
Hex = Wt
MUT Sequence (junction is in uppercase):
aaacccttgtagccactgagggacagctgtatacgcagtgctctcagaactgtgtctagcatacagtaggtgattaaacatgtgaatcctaagtcagttagagagtcactctaggatgaGCgaaatcttagtttccactcatagactttcccatgtgtttacatgctccctgctcccacagtcttagctaatttgcactctctctctctctctttctcctctc
This mutation is a 3508 bp deletion beginning at Chromosome 1 position 119,907,771 bp and ending after 119,911,278 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52175 | CCC TTT CTT TCT TGC AGT GG | Wild type Forward | A | |||
52176 | TCA CAA GTC TCC TCA CCT TGT C | Wild type Reverse | A | |||
52177 | TGC TCT CAG AAC TGT GTC TAG CAT | Mutant Forward | A | |||
52178 | TGG GAA AGT CTA TGA GTG GAA AC | Mutant Reverse | A | |||
52179 | Fluorophore-1 | CCT TGC CTT GAT CCT TGG A | Quencher-1 | WT Probe | ||
52180 | Fluorophore-2 | AGA GAG TCA CTC TAG GAT GAG CGA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52175 | 0.40 uM |
52176 | 0.40 uM |
52177 | 0.40 uM |
52178 | 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 |