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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
>chr7:126585346+126585475 130bp GTGCCTTCACTCTTCTTTCTTC CCTCAGTCACCTCTCCCAGT
Mut= 136 bp
Wt= 130 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GTACCCACTTATTCAAGCTAGGCCTTGTCGGGGTCCCTTAGCAGACGTCCAGATCCCTGATTCTTACCTGGGCTTTGTTAACAGTGTACTCCTGGGGAAGTGGTTAGACCTCCCCTGAGTGCCTTCACTCTTCTTTCTTCTCTTCTAGGACTCcttcagtgcgtttgtgtcctacctcgtaggagacacagtccccacagtagagaggcagacacaggcagctgaggaactgggagaggtgactgaggggaaggttgtaggagaggaagcccaggaggtactggagggacttagaagtggccagagtgagggggtagaagcacccgaagagactagaagatgccaagaaggaagtttagctggtgaacagacctgggggtggagagcagatagctctgcaaggccccaagcagaaaggcaggacactgggtcctggaaggcagctgaggatgccaggggccaggagccaagtgtcccactgaagcctgaggcagagcctgggactcacagagacaggagcagtaatacagcccaggagatctgggagcacggtgaagaggaagcaagcagtggggagcctctgagaacctgtgaacagaaggaagaagaggaggaggtggtcagagcagcagagtcagggatggccgagggggtggagtcacagccaacctggcacagcgagcctggggggaatgctggcactgaggggcagcatgtgacagaggacagcaaagagatagactgggtggccaaagacatggttgcagaaatcgaatggtttggtgccaaagggattgacaaggaagaggagaggatggtcccaatgagggatggtgaaagggcaagggcacaggggacacaatgccccggggccgagtctgaggaccaggccatgttgagtagagaggcctggacagtctcagacagggagggggctgatagcctaggagttcagggaacagaatacgggtcagacccaggagacaacttcccaggaactactgggagagtctgggtcctagaagaggctgacaagggagaccagcaggatgaggtcgacgagaagagagaggctgaagttaggtttccgatccagaccctggaggctgagagaactggagagatgactgaaggccacatagctgaagaggaggctatgggagaacaggaaacagagggcagctttgaggatgaggagaggcaagacttagccatcagggataatggagtgagtctagaagaagaggtacgggcagaagaatcctccagggagaaaaggaactcctgggccacagagcctacgctagtcctagacacggaggctaaagatgaacctgactgggaagactctccagaagtcagcactgaggagttgtttgtgggagagaggagtgaagccgcccagatgacaccagaggtattgagagtaaaggtcactgaaggacaggaccctgagctggtgagacattcccaggctttaactaaacaacttgaggaaggacagaagggtcaggaagagactagtggagctccagacctgagcccagagcgggtgctaagcttgaaggaatatcctgggcctgtggggtttgcaggccctgagctagaagcctggggaaactggagtaggggtgtggaccgcagaaatagccaggaggtaaaagcagatgctgaagcaggcaaggaacagactgcaaccgagcaggcagtggagatccgggctgaaggaggccaggaggcccaacagccagaggtcttcgggtcaggaggagaggaggcactgacctccgtagcacttaacccggagctggagggaagccaaggagcagaggcggggacagaagagtcagtggaggaatccaagcccacagaaaacgaggctgctgaggaggaggctgtagtgccttgggaggcagatggaacttgcaggaagaggaggctggaggaggtgactctgagcctgcaagacagtgaggacacagagaccagttatttggctgaggagattattgtgggtatcagggccgtggataccgaggaagggccaaaatgggaagctgggttggctccagaaacagagcttgggaaagcctggtgttccgagggcagaggggaggctgggagaggcacggagctggaggagactacagagaagcaaagtggacaggaagttggcttggtgggctcagcagagaaggtgtctggctatgatatccaagaaattgatgggactgaagagggggagcaggcagagatggagacatctgtaatggcagaagacataagggggacggatggtgtgactttaggctcccaggcagagagagcagaggggtccataacccccatggagactgaggggctcctaagagatcagatgctgttggaagaagaggctgggggagggcagtcgcgagagcagaaggtccacaactcagagggcgagatccaaacgctggacgactcttcagaccaggaggggcagcagacacaccaaatccccacagtagctgtgcctgggcccttggaatcggctgaggccacggcaggtgccccaggggatgttcACAGCAACTGGAATGAGGTGAGGGCTTCAGCAGGGCCCCGAGAGGGACAGGCAGAATCTGGAAGTCAGTTCTCCACAGTCCTCTATGTCCCTTATCCTGCAGGCCCTGCTCCCTGGGTCCCGCCTGGATGTCTCTGTCCCTCGGAGTCGTGTGCTCCTCTC
This mutation is a 2414 bp deletion beginning at Chromosome 7 position 126,585,381 bp and ending after 126,587,794 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 42625 | GTG CCT TCA CTC TTC TTT CTT C | Common | A | |||
| 42626 | CCT CAG TCA CCT CTC CCA GT | Wild type Reverse | A | |||
| 42627 | TGC AGG ATA AGG GAC ATA GAG G | Mutant Reverse | A | |||
| 42628 | Fluorophore-1 | AGA CAC AGG CAG CTG AGG A | Quencher-1 | WT Probe | ||
| 42629 | Fluorophore-2 | CTA GGA CTC ACA GCA ACT GGA A | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 42625 | 0.40 uM |
| 42626 | 0.40 uM |
| 42627 | 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.