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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:114762219-114762380 162bp TGCTGTTGTTTGTTCCGTTC GGATTCTGGGAAAGCAAATG
Mut = 170 bp
Wt = 162 bp
Fam = Mut
Hex = Wt
MUT Sequence (junction in uppercase):
ggaaatcctggcttagaggatatgaaatgtctacctctggcctccacacaagtacacacacatacatacatacataaacacacatgcatgcacacatacatatacatgtatacacacatacatacatgcacataCAcacacacacacccacacacacacaagcatttcttgaaataagcttcctgtgacaataataaaactcagaactcttacaaaaacgcaggcagtttttgtggtt
This mutation is a 5299 bp deletion beginning at Chromosome 9 position 114,758,536 bp and ending after 114,763,834 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51491 | GGG AAA TCC TGG CTT AGA GG | Mutant Forward | A | |||
53031 | AGA AAT GCT TGT GTG TGT GTG G | Mutant Reverse | A | |||
53034 | Fluorophore-1 | ATG TCT ACC TCT GGC CTC CAC A | Quencher-1 | MUT Probe | ||
54016 | TGC TGT TGT TTG TTC CGT TC | Wild type Forward | A | |||
54017 | GGA TTC TGG GAA AGC AAA TG | Wild type Reverse | A | |||
54018 | Fluorophore-2 | AGC TCT GCT CAT CCG AGA AGC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51491 | 0.40 uM |
53031 | 0.40 uM |
54016 | 0.40 uM |
54017 | 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 |