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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
Mutant= 113 bp
Wild Type = 121 bp
Sequence
Wt Sequence (deletions in lower case):
TTCTCCTTTCATACTTTCATAAACATTAGGAATTTCATCCCTGAATTTCAAAGTTCAAGATAATCTTAGCCCCTCAGagatacaacatacttctttcacaggatagctgaagacactgtggaggaaatggctttgtcagttttcaccttacccacagaaagtaggtgtccataggtctccagcatcacatcacggtacagagtccgctgaacaaggtctagctgtccccattcttcttgggtaaaggccacagccacatctttgaaagtcactggttcctgaaagagcatatatgatccttctcagccagaggccagcctttccgaattttctagggatagaatgaggctgatagcctttgaggagaggagacaggatgtaaaggacacactagAAGGGTTCAAAAATCTGGCCAGGTGTGGTGATTTACTTTAATAAAGCACTTGGGAGGCAGAGGTAGGTGGATC
This mutation is a 317 bp deletion of Chr7:12,489,454-12,489,770 (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 80038 | CCT CCC AAG TGC TTT ATT AA | Common | A | |||
| 80512 | TCT AGG GAT AGA ATG AGG CT | Wild type Forward | A | |||
| 80513 | ACA TTA GGA ATT TCA TCC CTG A | Mutant Forward | A | |||
| 80514 | Fluorophore-1 | TTT GAG GAG AGG AGA CAG G | Quencher-1 | WT Probe | ||
| 80515 | Fluorophore-2 | AGA TAA TCT TAG CCC CTC AGA AGG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 80038 | 0.40 uM |
| 80512 | 0.40 uM |
| 80513 | 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.