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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.
Mutant= 137 bp
Wild Type = 148 bp
>chr2:24952437+24952584 148bp GTTGTTGATCAGGATGTGACC TGAGAAGAAGCACAGAGCAGAG
Wt Sequence: gttgttgatcaggatgtgacccctGtacttttggggtcctaggagcaggatatcatctgtacttctggcatttttctgagctggatttgaaatatgctgagagttaaaggctttccaacagatgcactctgctctgtgcttcttctca
Mutant Sequence: gttgttgatcaggatgtgacccctGagaaggatccatgggtctcatctcatcaaaatcccactagttgtttaaagtgatatgtacattagtcactcctttgGgtgagtcataggttacatccttagaaacggggtca
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
35386 | GTT GTT GAT CAG GAT GTG ACC | Common | A | |||
35387 | TGA CCC CGT TTC TAA GGA TG | Mutant Reverse | A | |||
35388 | TGA GAA GAA GCA CAG AGC AGA G | Wild type Reverse | A | |||
35389 | Fluorophore-1 | ACT CCT TTG GGT GAG TCA TAG GT | Quencher-1 | MUT Probe | ||
35390 | Fluorophore-2 | TTT TGG GGT CCT AGG AGC A | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
35386 | 0.40 uM |
35387 | 0.40 uM |
35388 | 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 |