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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 = A/A
Heterozygote = G/A
Wild type = G/G
X-linked
>chrX:72819935-72820023 89bp TTACAATCCAGGAGGGTTGG CCTCTCTGATTTGATAACCTCTTC
Sequence
ATGCCATTAATGTGGAGAAAGCCTTCACAGACCTGACAAGAGACATATATGAGCTGGTTAAAAGGGGGGAGATTACAATCCAGGAGGGTTGGGAAGGGGTGAAGAGT(g/a)GATTTGTACCAAA(t/c)GTTGTTCATTCTTCAGAAGAGGTTATCAAATCAGAGAGGAGATGTTTGTGCTAGTTAACCCTTCCAT
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 43632 | TTA CAA TCC AGG AGG GTT GG | Forward | A | |||
| 43633 | CCT CTC TGA TTT GAT AAC CTC TTC | Reverse | A | |||
| 43634 | Fluorophore-1 | AGG GGT GAA GAG TGG ATT TGT ACC | Quencher-1 | WT Probe | ||
| 43635 | Fluorophore-2 | AGG GGT GAA GAG TAG ATT TGT ACC AA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 43632 | 0.40 uM |
| 43633 | 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.