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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.
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
Mutant= 225 bp
Wild Type = 113 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 15244 | TTG GGA AGA CAA TAG CAG GC | Mutant Forward | A | |||
| 19912 | CGC TGT CCA CAG TGG TTG T | Common | A | |||
| 22481 | ATC TGG TCA TGG GAT TGC TG | Wild type Forward | A | |||
| 22482 | ACA CAG CAC ATC TAC CGA AG | Wild type Reverse | A | |||
| 22483 | Fluorophore-1 | CTC GCA GAA GAA GGA GCC GTA CT | Quencher-1 | WT Probe | ||
| 22485 | Fluorophore-2 | TTC CGC CTC AGA AGC CAT AGA GC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 15244 | 0.40 uM |
| 19912 | 0.40 uM |
| 22481 | 0.40 uM |
| 22482 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| DNA |
Cycling
| 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 |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.