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Notes
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 = 66 bp and 90 bp
Heterozygote = 66 bp and 90 bp and 156 bp
Wild type = 156 bp
Heterozygote = 66 bp and 90 bp and 156 bp
Wild type = 156 bp
Separated by gel electrophoresis on a 5% agarose gel.**
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR1123 | CAC AGT ATT TGG CAA CGT GC | Common | ||||
| oIMR1124 | TAA CCC ATA ACC TCG TTG GC | Common |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| AB PCR BufferII | 1.00 X |
| MgCl2 | 2.50 mM |
| dNTP | 0.20 mM |
| oIMR1123 | 1.00 uM |
| oIMR1124 | 1.00 uM |
| DNA Loading Dye | 0.69 mM |
| Taq DNA Polymerase | 0.03 U/ul |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 55.0 | -- | |
| 4 | 72.0 | -- | repeat steps 2-4 for 35 cycles |
| 5 | 72.0 | -- | |
| 6 | 10.0 | -- | hold** |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
Reaction B
| Component | Final Concentration |
|---|---|
| AB PCR BufferII | X |
| ddH2O | |
| BSA | mg/ml |
| Nhe I |
Strains Using This Protocol
This is the only strain that uses this protocol.