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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= 106 bp
Wild Type = 110 bp
Wt Sequence: ACTGTGAGATGCAGGACAGCGATGCCGTTCCGGTGGGGATCCCtCCTCCCGCTTCTCTGGCCGCTAACCTCGCAGGGCCACCGTGCGCTCCCGAAGGCCCTATGGCAGCC
Mutant Sequence: ACTGTGAGATGCAGGACAGCGATGCCGTTCCGGTGGGGGATCCCgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagc
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
14989 | GCT GCA AGG CGA TTA AGT TG | Mutant Reverse | A | |||
33270 | ACT GTG AGA TGC AGG ACA GC | Common | A | |||
33271 | GGC TGC CAT AGG GCC TTC | Wild type Reverse | A | |||
33272 | Fluorophore-1 | TCG TGA CTG GGA AAA CCC T | Quencher-1 | MUT Probe | ||
33273 | Fluorophore-2 | AAC CTC GCA GGG CCA C | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
14989 | 0.40 uM |
33270 | 0.40 uM |
33271 | 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 |