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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.
Mut= 109 bp
Wt= 105 bp
Wt Sequence:
Mutant Sequence:
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
34381 | AGG TAA AAG TTT ATG TCA TGT GGT TG | Common | A | |||
34382 | Fluorophore-1 | CAT GGA GAT AGA AAC ATC TAA CTA GGA | Quencher-1 | WT Probe | ||
34383 | Fluorophore-2 | TTG ACA TAG AAG TTC ACA TTA TCT TGA | Quencher-2 | MUT Probe | ||
34384 | ATG ACA GGC ATG TGT CTC ACC | Wild type Reverse | A | |||
34385 | TTC CAA ACT TGA TGA AAA TGT CTC | Mutant Reverse | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
34381 | 0.40 uM |
34384 | 0.40 uM |
34385 | 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 |