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Notes
Taqman Probe/Endpoint protocols use a different analysis than qPCR. Please see your equipment manual for more information about Taqman endpoint analysis setup.
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
Sequence
Large deletion: Mutant sequence with junction in uppercase
gtaaggaagggatgataagtatcagggtcttcacccccagtaatcataatgatgttggtctgagttctctcttagcttctgtcATtctttttgatagacatttcaagtataagtatcaatatagtactattcctaacatgatttctgtagatctttatgggataaaactggatgcatcaaatat
Mutation details: This mutation is a 3,117 bp deletion of ChrX:89,752,397-89,755,513 (GRCm38/mm10)
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 77407 | TCA ATG CAA TTC AGT ACC AA | Wild type Forward | A | |||
| 77408 | AGA TCT ACA GAA ATC ATG TTA GGA | Common | A | |||
| 77409 | GAT AAG TAT CAG GGT CTT CAC C | Mutant Forward | A | |||
| 77410 | Fluorophore-1 | TTC ATG AAT AAA GGA CTA AGC T | Quencher-1 | WT Probe | ||
| 77411 | Fluorophore-2 | AGT TCT CTC TTA GCT TCT GTC ATT CT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 77407 | 0.40 uM |
| 77408 | 0.40 uM |
| 77409 | 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.