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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 = --/--
Heterozygote = AC/--
Wild type = AC/AC
Sequence
ctgcagACCAAGCTCTTGACCCAGTATGTGCTGAGTCTGGCCAAGTACGACCAGAac/--TATGATATCCGGGATCGAG
CCCGCTTCACCCGGCAGCTCATTGTTCCCTCTGAGCAGGGTGGTGCCCTCAGTCGCCATGCCAAGAAACTCTTC
CTGGCCCCTAAGCCAGCCCCTATCTT
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 18421 | CTG CAG ACC AAG CTC TTG AC | Forward | A | |||
| 18422 | AAG ATA GGG GCT GGC TTA GG | Reverse | A | |||
| 18693 | Fluorophore-1 | GTA CGA CCA GAA CTA TGA TAT CCG | Quencher-1 | WT Probe | ||
| 18695 | Fluorophore-2 | AGT ACG ACC AGA TAT GAT ATC CGG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 18421 | 0.40 uM |
| 18422 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | |
| 4 | -- | repeat steps 2-3 for 40 cycles | |
| 5 | 40.0 | -- | Forever |
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.