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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
>chr11:62119483+62119590 108bp AAACCCACCCAGATTTCTGTA ACACAGGTCACCCCTGACG
Mut= 142 bp
Wt= 108 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
AGAAACCCTATTCAAAACAGATTTTGAGCATGTCACTGCAGACACCCCATCTCAGCCCCTGTCAGCCGCTAGCAACCCTTTTAAGAGCTCCAAGGGCTCCCCTACTGGGAACTCCCCAAACAATTCAAATGAGTTGTCCTTGCCGCTCTTACTGAGAAGatacctagaggaggaagaattggatgaagagtgacagccatagcatcatctatgccatatatggaagacatttgagtttcattataaaattttttttctttcttttttgtttcattttttctttcatcttttgtctttagggactattccaacaactaaacggacaggcattccagctccacgggagctctcagtaactatctccagagagaggtctgtgccacgtggtccctccagctcaaagaagttaggctccagcccaacttcttcctgcaatcccactcctaccaagcatctgaggaccacccctgcaaaacctaagcaagagcatgaaggtgcagagaaggctgtgctcgagtcccaggtacgagaacttctggcagaagccaaaaccaaagacagtgaaatcaacaggcttcgaagtgaactaaaaaaatgcaaagagaggtgggccctgagcactgaggatgccaatgcttcagacccaagtgcagagggaacagcatccccagaaagtgatgcacagcctttgataagaaccctggaggagaagaacaagacctttcagaaagagcttgctgacctagaggaagaaaacagggccttgaaggagaaactgacttaccttgagcagtcaccaaattcagaaggggcagcaagtcacactggtgacagcagctgcccaacatctataacccacgagtccagttttggaagcccagttggaaatgagttgtctagtgaaacagatgagtacagaagaaccacacatgggagtgccttgcggacatcagggtcttcaagcagtgatgtcaccaaagcctctctgtcaccggacgcctcagattttgagcatatcactgcagacaccccgtctcggcccctgtcagccactagcaacccttttaagagctccaagggctcccctactgggagctccccaaacaatgcaagtgagctgtccttggcatctctcacggagaagatacagaagatggaggagaatcagcacagcactgcagaagagctgcaggctactctgcaggaattatcagaccagcagcagatggtgcaggaactgacagccgagaacgagaagctagtggatgagaagaccattctggagacctccttccaccagcaccgagagagggcagaacagctgagtcaggaaaatgagaagctgatcaaccttttacaggagcgagtgaagaatgaggaacccagtgcccagggagggaaggtccttgagctggagcagaagtgcacggatattctcgagaagagccgctttgaaagagagaagctgctcaacattcagcagcagctgacctgtagcctacggaaggttgaggaagaaaaccaaggcgctatagacatgattaagcacctgaaggaagaaaatgagaagctgaatgggttcctagaacatgagcggtgtaataatagtgtgatggccaaaactctggaggaatgtcgagttaccttggaaggcttgaaaatggagaatgggtccttgaaggctcttttggaggctgataagcaaaaagccatagaggccagcagtactgtggggcagacagcagagaacttcgaggttcaggagatgctgaaagtggctcgagctgagaaagatcagttgcagctgtcttgcacagagctcagacaagaattgctaaaggcaaatggtgaaattaaacatgtttccagcctgctagctaaggtaagaagacagttctcaactagtatttgttgacatttctctttgagaccttaacattgtgaaccaaagagaatcaattattcaactgtatttgtgaatctaagccttctgatttggaaatctattagaatttaggcaggttagctctggggattccaaattttaaaacaaaacaaaacacaaaacaacaacaacaaaaaaaacccacccagatttctgtacagatagataaaaagattagtggaaatactgtaaattacatcagatgatatggtgtagTGATGAAGACCGTCAGGGGTGACCTGTGTCTTGGTGGTAGAATACTTTCATAGCCTCCTTGAGACTTTAGATTTGATCCCTAGCTCCACACAAAAATAGGATAAAATAAGATTTAGAACCATCCTGTATACATTGATGGACTTTTTGATACAT
This mutation is a 2002 bp deletion beginning at Chromosome 11 position 62,117,560 bp and ending after 62,119,561 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 47023 | AAA CCC ACC CAG ATT TCT GTA | Wild type Forward | A | |||
| 47024 | ACA CAG GTC ACC CCT GAC G | Common | A | |||
| 47027 | CCA TCT CAG CCC CTG TCA | Mutant Forward | A | |||
| 47028 | Fluorophore-1 | AGT GGA AAT ACT GTA AAT TAC ATC AGA TG | Quencher-1 | WT Probe | ||
| 47029 | Fluorophore-2 | AGA GCT CCA AGG GCT CCC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 47023 | 0.40 uM |
| 47024 | 0.40 uM |
| 47027 | 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.