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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= 121 bp
Wt= 116 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
31562 | CAT CTT CCC CTT GTC TCC TG | Common | A | |||
31563 | AAT CCC CGC ATG CTT TTC | Wild type Reverse | A | |||
31564 | GGA ATC AGT CTT CCC AAA TTA CTG | Mutant Reverse | A | |||
31565 | Fluorophore-1 | CTT GCA CTG CCC ATC ATT TA | Quencher-1 | WT Probe | ||
31566 | Fluorophore-2 | TGT GGG GAG GAC TCT CAG GT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
31562 | 0.40 uM |
31563 | 0.40 uM |
31564 | 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 |