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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= 111 bp
Wild Type = 108 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
32221 | TCA TGC ATT TCC GAT AAG ACA | Mutant Forward | A | |||
32222 | Fluorophore-1 | TTT CTG TGG CTC TTC AAC TCT TT | Quencher-1 | MUT Probe | ||
32223 | ATG GAC CAG TGA GAA ACG TAT G | Wild type Forward | A | |||
32224 | Fluorophore-2 | ATT GAC TTT CGG CAG CAT AG | Quencher-2 | WT Probe | ||
32225 | GCA TCA CTT TAT ACT AAC TGA GAG AGG | Common | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
32221 | 0.40 uM |
32223 | 0.40 uM |
32225 | 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 |