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Stock No: 004068
Protocol 20541: Probe Assay - Idua<tm1Clk> Probe
Version 1.0
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= 75 bp
Wild Type = 66 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 23675 | CCT GGG GAT TCC TTC CAC | Common | A | |||
| 23676 | GGC ACA GTG ACC CAG GAG | Wild type Reverse | A | |||
| 30625 | GGC TCT ATG GCT TCT GAG G | Mutant Reverse | A | |||
| 30628 | Fluorophore-1 | CTG CCA AGG TCA CCA ATG T | Quencher-1 | WT Probe | ||
| 30629 | Fluorophore-2 | CCT GCC AAG GTC ACG AG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 23675 | 0.40 uM |
| 23676 | 0.40 uM |
| 30625 | 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.