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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.
X-Linked
Mutant= x bp
Wild Type = y bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
31188 | TAG TGA CCT AGG ATT AGA TCC TTT CAG | Wild type Forward | A | |||
31189 | TGA AAA GTC ATC CAT TCT CTT TTA | Mutant Forward | A | |||
31190 | Fluorophore-1 | AGC CAG CGT TTG TCA TCT TT | Quencher-1 | MUT Probe | ||
31191 | Fluorophore-2 | TCT TAT TCT TTA GCA AGG TTA TAG TGG | Quencher-2 | WT Probe | ||
31192 | TGG AAT GGT ATT ATT TTG CCA CAG | Common | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
31188 | 0.40 uM |
31189 | 0.40 uM |
31192 | 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 |