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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
tagatgtgcagtatttgtgctgaatgtaaatgtgattcttatagaggttatagtcaaataggcaaactgttagatatgtttaatcttcaggatgcatattgccactggaaaataacatcacttagccataatgtcatgttctagagaattatattacagttcctatagaaataaaatctttaacccagtcaaatgtcttacacttgttagaATctcgggtgacaggctgatgactgatggcattgatgacactatcgctgagctctgacatttccagtacagtgcttctcagcactcctaatgctgcagccctttcaggaagt
This mutation is a 2,192 bp deletion of Chr7:55,798,614-55,800,805 (GRCm38/mm10)
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 76040 | AGG AAA TCT CTT CAC TAA GCC | Wild type Forward | A | |||
| 76042 | AGT GTG TAT GCT CAT CAT GG | Wild type Reverse | A | |||
| 76043 | CCA TAA TGT CAT GTT CTA GAG AA | Mutant Forward | A | |||
| 76044 | ATC AAT GCC ATC AGT CAT CA | Mutant Reverse | A | |||
| 76045 | Fluorophore-1 | CTG CAT GCT GAG AAG ACG | Quencher-1 | WT Probe | ||
| 76046 | Fluorophore-2 | CAC TTG TTA GAA TCT CGG GTG ACA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 76040 | 0.40 uM |
| 76042 | 0.40 uM |
| 76043 | 0.40 uM |
| 76044 | 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.