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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.
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR1544 | CAC GTG GGC TCC AGC ATT | |||||
| oIMR3580 | TCA CCA GTC ATT TCT GCC TTT G | |||||
| oIMR4082 | TGA AGG GCG AGA TCC ACA A | |||||
| oIMR4083 | TGG ACT TGA ACT CCA CCA GGT A | |||||
| TmoIMR0067 | Fluorophore-1 | CTG AAG CTG AAG GAC GGC GGC C | Quencher-1 | |||
| TmoIMR0107 | Fluorophore-2 | CCA ATG GTC GGG CAC TGC TCA A | Quencher-2 |
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.91 |
| ddH2O | |
| oIMR4082 | 0.43 |
| oIMR4083 | 0.43 |
| oIMR1544 | 0.00 |
| oIMR3580 | 0.00 |
| TmoIMR0067 | 1.27 |
| TmoIMR0107 | 0.00 |
| DNA |
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 65.0 | -- | -0.5 C per cycle decrease |
| 4 | 68.0 | -- | |
| 5 | -- | repeat steps 2-4 for 10 cycles | |
| 6 | 94.0 | -- | |
| 7 | 60.0 | -- | |
| 8 | 72.0 | -- | |
| 9 | -- | repeat steps 6-8 for 28 cycles | |
| 10 | 72.0 | -- | |
| 11 | 10.0 | -- | hold |
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