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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 = aaatgttgttc/-----------
Wild type = aaatgttgttc
Heterozygote = aaatgttgttc/-----------
Wild type = aaatgttgttc
X-linked
>chrX:72819926-72820019 94bp AATCCAGGAGGGTTGGGAAG CAAACATCTCCTCTCTGATTTGA
Sequence
TGAAGTACATTGAGACGTCAGCCCGAGATGCCATTAATGTGGAGAAAGCCTTCACAGACCTGACAAGAGACATATATGAGCTGGTTAAAAGGGGGGAGATTACAATCCAGGAGGGTTGGGAAGGGGTGAAGAGTGGATTTGTACC[aaatgttgttc]ATTCTTCAGAAGAGGTTATCAAATCAGAGAGGAGATGTTTGTGCTAGTTAACCCTTCCATTTTCAAAACATACTCTCCCACTTGAACTTAAAAGTTATCAAAATAAATAGAATCCTTATGTGACTGAAGAGAACTCAACGTTTGGATAATAAATGAGCCT
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 43636 | AAT CCA GGA GGG TTG GGA AG | Forward | A | |||
| 43637 | CAA ACA TCT CCT CTC TGA TTT GA | Reverse | A | |||
| 43638 | Fluorophore-1 | TTG TAC CAA ATG TTG TTC ATT CTT C | Quencher-1 | WT Probe | ||
| 43639 | Fluorophore-2 | TGG ATT TGT ACC ATT CTT CAG AAG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 43636 | 0.40 uM |
| 43637 | 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.