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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 sequence, including junction:
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
19764 | CTT TAC GGT ATC GCC GCT C | Mutant Forward | A | |||
19765 | Fluorophore-1 | CGC ATC GCC TTC TAT CGC CTT CT | Quencher-1 | MUT Probe | ||
19766 | AGG ATC TTC TCG CTG TTT GTC | Common | A | |||
19767 | CGT TAT AAG CAG GTG TGG GTC | Wild type Forward | A | |||
19768 | Fluorophore-2 | TCT GCA CAG GTG AAG GAT GTC ATG AAA | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
19764 | 0.40 uM |
19766 | 0.40 uM |
19767 | 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 |