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Notes
Incubate 8 of PCR product with 4 µl restriction enzyme master mix for a minimum of 4 hours at 37 degrees C prior to running on gel.
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 = 108 bp
Heterozygote = 108 bp and 88 bp and 20 bp
Wild type = 88 bp and 20 bp
Heterozygote = 108 bp and 88 bp and 20 bp
Wild type = 88 bp and 20 bp
Add DNA Loading Dye* to digested products and separate on a 3% agarose gel.**
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR1110 | CAA GAC AAT ATT CCT GGT GCC | Reverse | ||||
| oIMR1163 | CAA TTT TGA GGA ATC TAA GGC C | Forward |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| AB PCR BufferII | 1.00 X |
| MgCl2 | 2.00 mM |
| dNTP | 0.20 mM |
| oIMR1163 | 1.00 uM |
| oIMR1110 | 1.00 uM |
| DNA Loading Dye | 0.69 |
| 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 |
|---|---|
| Rest Enzyme Buffer | 1.00 |
| ddH2O | |
| Stu I | 0.83 U/ul |
| PCR product |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 37.0 | -- | 2-6 hours |
| 2 | 10.0 | -- | hold |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.