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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
Apc = ~340bp
heteroduplex (occasional artifact) = ~1000 bp
wildtype = ~600bp
heteroduplex (occasional artifact) = ~1000 bp
wildtype = ~600bp
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 |
|---|---|---|---|---|---|---|
| oIMR0033 | GCC ATC CCT TCA CGT TAG | Wild type | ||||
| oIMR0034 | TTC CAC TTT GGC ATA AGG C | Common | ||||
| oIMR0758 | TTC TGA GAA AGA CAG AAG TTA | Mutant |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| AB PCR BufferII | 1.00 X |
| MgCl2 | 2.50 mM |
| dNTP | 0.20 mM |
| oIMR0033 | 1.00 uM |
| oIMR0034 | 1.00 uM |
| oIMR0758 | 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 | -- | Go to step 2-4, 35 times |
| 5 | 72.0 | -- | |
| 6 | 10.0 | -- |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
Strains Using This Protocol
This is the only strain that uses this protocol.