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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 = 350 bp and 180 bp
Heterozygote = 180 bp, 350 bp and 530 bp
Wild type = 530 bp
Heterozygote = 180 bp, 350 bp and 530 bp
Wild type = 530 bp
Separated by gel electrophoresis on a 1.5% agarose gel.**
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR1586 | AGG CAG GAA GAT CAC GTG TTC AAG TAC | |||||
| oIMR1587 | CAC ACG CAC ACT CTG ACA TGC ACA GGC |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| KAPA Rxn Buffer Contains 1.5 mM Mg2+ final concentration. | 1.00 X |
| dNTP | 0.20 mM |
| oIMR1586 | 1.00 uM |
| oIMR1587 | 1.00 uM |
| DNA Loading Dye | 0.69 mM |
| KAPATaq DNA Polymera No proofreading function. 1 error per 40000 nucleotides. Initial denature 2 min. | 0.04 U/ul |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 69.0 | -- | |
| 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 |
|---|---|
| ddH2O | 0.00 |
| NEB Buffer 3 | 0.86 X |
| BSA | 0.09 |
| BsteII | |
| PCR product |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 60.0 | -- | USE MJ #7 |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.