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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= 133 bp
Wild Type = 156 bp
Wt Sequence: CTAAAGCCCACTTATAACCCAGCATACAAATAgatcTTAACAGTTTATTATTAACTTTTCAGGTCCTCATTTATTGAAATTTAAAAAT
Mutant Sequence: AAAGCCCACTTATAACCCAGCATACAAATAGATCccaagcgctctgaagttcctatactttctagagaataggaacttcggaataggaac
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
32191 | Fluorophore-1 | CCA GCA TAC AAA TAG ATC TTA ACA GT | Quencher-1 | WT Probe | ||
33316 | GCT CCT TAT GAA GAA AAC CAG CA | Common | A | |||
33317 | GCT TTG AAG TTC CTA TTC CGA AG | Mutant Reverse | A | |||
33318 | CTG GAA CCC TGT GCC ATC T | Wild type Reverse | A | |||
33319 | Fluorophore-2 | CAA GCG CTC TGA AGT TCC TAT ACT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
33316 | 0.40 uM |
33317 | 0.40 uM |
33318 | 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 |