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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr14:69799840-69799955 116bp GCAGCAAGGAGTGTGCTCTT GGGAGGACAGGACTGGTACA
Mutant= 88 bp
Wild Type = 116 bp
Wt Sequence: gcagcaaggagtgtgctcttttaaGgtctcccctgcttccgctcagcgtggagattgccagtgatttcatgtgggcagcaggttgtacaagacacttgtaccagtcctgtcctccc
Mut Sequence: gcagcaaggagtgtgctcttttaaGGcaggtgggtagtcagggccctgtgactgcttggggccgattctccagcatatccactgtgtc
A 3821 bp deletion beginning at Chromosome 14 position 69,796,110 bp and ending after 69,799,930 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
66275 | GCA GCA AGG AGT GTG CTC TT | Common | A | |||
66276 | GAC ACA GTG GAT ATG CTG GAG A | Mutant Reverse | A | |||
66277 | GGG AGG ACA GGA CTG GTA CA | Wild type Reverse | A | |||
66278 | Fluorophore-1 | AGG CAG GTG GGT AGT CAG G | Quencher-1 | MUT Probe | ||
66279 | Fluorophore-2 | CAG CGT GGA GAT TGC CAG | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
66275 | 0.40 uM |
66276 | 0.40 uM |
66277 | 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 |