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Notes
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.
Alt1 - Trying a smaller Tg probe. DSK 2/27/23
The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX).
To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility.
Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.
Expected Results
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 65586 | Fluorophore-1 | ACT AGG CAG TTA CAC TGG AGG TGG | Quencher-1 | Tg Probe | ||
| oIMR1544 | CAC GTG GGC TCC AGC ATT | Internal Positive Control Forward | A | |||
| oIMR1799 | GGA ATG GAA AGG AAA GTG AAT ATT TG | Transgene | A | |||
| oIMR1800 | GCT GTC TCC TAG CAA CGA CTT CT | Transgene | A | |||
| oIMR3580 | TCA CCA GTC ATT TCT GCC TTT G | Internal Positive Control Reverse | A | |||
| TmoIMR0105 | Fluorophore-2 | CCA ATG GTC GGG CAC TGC TCA A | Quencher-2 | IC Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| oIMR1544 | 0.40 uM |
| oIMR1799 | 0.40 uM |
| oIMR1800 | 0.40 uM |
| oIMR3580 | 0.40 uM |
| Tg Probe | 0.15 uM |
| IC Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | repeat steps 2-3 for 40 cycles |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.