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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 = ~110 bp and 58 bp
Heterozygote = 20 bp, 53 bp, 58 bp, 90 bp and ~110 bp
Wild type = 90 bp, 53 bp and 20 bp
Heterozygote = 20 bp, 53 bp, 58 bp, 90 bp and ~110 bp
Wild type = 90 bp, 53 bp and 20 bp
Separate digested PCR product on a 5% agarose gel.**
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR1292 | GGC CCA GCA GGC AGC CCA AG | Common | ||||
| oIMR1293 | AGG GAT CTC CTC AAA GGC TTC | Common |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| AB PCR BufferII | 1.00 X |
| MgCl2 | 2.50 mM |
| dNTP | 0.20 mM |
| oIMR1292 | 1.00 uM |
| oIMR1293 | 1.00 uM |
| DNA Loading Dye | 0.69 mM |
| Taq DNA Polymerase | 0.02 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 | |
| BseDI |