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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:37302784-37302897 114bp AAAGAGGAGGATTGAAAAGT AGCCCAAACATGGTGATTTC
Mut= 102 bp
Wt= 114 bp
Fam=Mut
Hex=Wt
Mut Sequence:
tctgttagacttctttgcagtatctaaagacgtactgttcttaaagtttCGgtaatggaatgcttgctaacaccaaaggctagaaatcaccatgtttgggct
This mutation is a 13,775 bp deletion beginning at Chromosome 9 position 37,302,836 bp and ending after 37,316,610 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48073 | AAA GAG GAG GAT TGA AAA GT | Wild type Forward | A | Wt F | ||
48074 | AGC CCA AAC ATG GTG ATT TC | Common | A | Common | ||
48075 | TCT GTT AGA CTT CTT TGC AGT ATC T | Mutant Forward | A | Mut F | ||
48076 | Fluorophore-1 | CTC ATT CTG AGA CTG AAG TAA CCA GTT | Quencher-1 | WT Probe | ||
48077 | Fluorophore-2 | CTT AAA GTT TCG GTA ATG GAA TGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48073 | 0.40 uM |
48074 | 0.40 uM |
48075 | 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 |