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Notes
Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
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= 116 bp
Wild Type = 126 bp
Sequence
Wt Sequence (deletions in lower case):
AGCTCCGGCCTGGGTTTTCTGTCCAAATCAGACCCTTGATGTCCAGACTCTTAAGGCTAGCCTTTGTATTTCAAGACATAGTTGACAAGATCATCACGATATAAGGACTTTATATCATGTGGTGTCATGGAATAATTTCCATTGAAAAGTTGTGTGTTGTGTTGCTGTGGACCCTGACCCTCCattttctgtaaggcgggcatagttgcacctccttcgtatcagttgtaaatgagacagctggagtaggacagatgcctgagcttgacacagagaccagggcatatggcaactgggaggcctgctttgtgtgtgcagtggcctctgttcacacagagtttcccctggcctccagggcaggagctctacctgctggagccctgtgcatgtctgtctgctggagagcagaagtggggaacccatttggcttcaaagattttttttcccctgttttaagggtgtcagcattgaagccatgaatgagagtaaaatggcgtcctcggagttaagctcggggcctgtggaaaaagctgccaagccgttgccatttaaggatcccaactttgtggtaagcatatgaggtggtatatcaatacagggtgctgtaccttagcttctgatctagctgggcctgaggtggctctggagtaccctcttcctcagtcccatttgccaccttctcctgtgggctgttggagacggtagtcgggggttgggagtcgggggttggggggagagagtggcagttggcggaaaagggatttgggactggacaggaaaggccctttgtgatctcccagtgtagcagaactttgaggaggcatcttttccatgattgtctccctgtgcttttttactggctttaaataattttaaaagatgcttgaaatatacaactaaaataggagtagtcctcataaccctttatagccctttgattcatgaaggttatattaagtagccagttacaaattataactttaaaggtttattgtttttattaatgagttttggcaatgtactgtagccagggtctgcaaatatgttagtagtcttttccacttcatgcttttaatctttatagcacagaaagaaatttccagaaggcttgccacaccatgattgaagcttatttgagctttccaggaccactggtgattatgaggctcagtgacacacccttcgagggggtttagagatgtagtctcgagattaaatccaactgtgtggtaaacttattttattttattttttttattttttatttttttttggtttttcgagacagggtttctctgtatagccctggctgtcctggaactcactttgtagaccaggctggcctcgaactcagaaatccacctgcctctgcctcccaagtgctgggattaaaggcgtgcgccaccacgcccggccaacttattttaaaggaatgagatcataagtgaattcaactagattggatcacataaagaatttttcctgtgtgcatgctgaaaaatagcatttacttctgtcccatcaccagcaaacttgtgggtttgaagagcccccaaactaggtcctttttgtttgcttgtttattttcatcttttgttttttgttttttgataaaggtcacctgtagcccaggctggcctcaaactccctactcttctgcttctacctcctgagtgctggcattgtagtgacatccaccacgcatggctaaaaactatgttctttcaaacaaagatttaagtgtaggtgaggtacagatagttactgatggaccgagaggtgcctgacaaccctaaagggcaggggcagggcgtcacgcttctccaggcgcgtcttccactgacttctcttcaaaccttcctttaattcaagcactctggccatggcggcgctgtagctggcaagaagaacagaacctggaagaacctcaaacagatcctcgctgctgaaagggcgttgccatggcaactgaacgatcctaactgtgagctgtctagagccttccatgtgtttgtaatgcagacaggaacttccttacaacaagcctacgttatttgctcgtgtttgtgttacttcagggaaatattcttagcctgagacccgcacagtggcacccatccagggatagtctgcatgtattccctttccactgagttggtgttgaggctgtttgtttgtcacggctgtgtgggttttagtgtcattatttaaaatttcagtctggagaaataggtcccgccgagataatgttgatgcttctattaagcttcccattttaaagtctactctttaagtttgttttcacaaacaacgtagcccacctaatgtgagaactggaaagtctccagcgtgcaggtaccagccactctctcactctctcctcccactgtagcatgtcctggcctgggcagaatccgttggtctactcaagtgcattggaaactttaggttccaagacctctccacctcctgccccgctgttcgtctttcatgggggtgactcagagggtgacaagttagtcatctgggtgtggactgcttgagtctgggtgtgtggatccagctgatggctgtgtggcctttcttcattgcctgttccactcagctggagcctgtgaagtagcctgcgggtagatgaacctggccgttccactggaggaacgtgtctctggtagcaaggcaggaccccagctgaggcccacacttagacatgctgggctggcttttgagccgtctctttttaagtgtgtaatcacaaaggaatatttcaggactgctgtcatgtgttagcttagagtaaaggagggggagtcgggatagttagaaggttatttggtgatcattttcttttctttcttttagactttagtattgatgctcctccatccttcaagccagctaagaagtattctgatatttcaggccttcttgtgagtataattacatctgtctatattagcactaaatagatttaagttatgtctctctagACAGATGGCTCTGCCTGGAGTCCATGCTGTGACCTGAGTGTCTGTGTGGGTAACCTGGAGGGGCCATTGCTCCAGTAAGCAAGACACAGAATTTTTACTTTTTTCCCTGTGATAGTTAACTGTATGCGACAACATGAATGAACCACCTTTCTGTTTTACTCTTGAGGTTTTAAAATACTCATATACAATTGGTGGTAGTTTAAAAACAAAT
This mutation is a 2807 bp deletion beginning at Chromosome 18 position 24,111,655 bp and ending after 24,114,461 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48303 | CCT CCC ACT GTA GCA TGT CC | Wild type Forward | A | |||
| 48304 | CTG AGT CAC CCC CAT GAA AG | Wild type Reverse | A | |||
| 48305 | TGT GTG TTG TGT TGC TGT GG | Mutant Forward | A | |||
| 48306 | CTT GCT TAC TGG AGC AAT GG | Mutant Reverse | A | |||
| 48307 | Fluorophore-1 | CAA GTG CAT TGG AAA CTT TAG GTT | Quencher-1 | WT Probe | ||
| 48308 | Fluorophore-2 | TGA CCC TCC ACA GAT GGC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48303 | 0.40 uM |
| 48304 | 0.40 uM |
| 48305 | 0.40 uM |
| 48306 | 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.