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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
>chr17:24850316-24850451 136bp CACGCCCTCAATCCATCAG GCTAGGGCAGCTAGGGAACA
Mut= 106 bp
Wt= 136bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
ACTTACCCAGACCACGGCGCGCGCAGGTGGCTCCCAGCGCTGACAGGCTCCGGAGACACAGCGACCCGCGACCCAGCACCATGACCCACGGGGTGCACAGCCGGGTCGCGGAGTCCGCAACACCAGCGTCGTGGGCATCAACACTGGCGTTGGCGACTGCAACCAATCAGTGTCGTCCTCCTACTGCTCCCAACCAACCAACTCCTGCTCTTTACCAAAATGCCACGCCCTCAATCCATCAGTGACCGCCcatagagttcacagccaaccagtgcctgtctcgcaccgccgcgccgcccctagccaatcagtagtagctcctgccccccccccccaaaatgttccctagctgccctagcagccttggcctgtagactcggtctgcaagtgggtgttggaggaggctgagcgtgagcctcctcgtctctctggggcagtcgcctgcacgcagagacctttcgctgacctcagcgtcccgctgctgcgcaaggaagggcggggccgctccgacctggacagcgtccgaagccagtcctctggaggccgccgtagtccgggggagtcggtggtcacatgacccaaagctgtactatggcttccaccaaaccgctgtctcgtttctgggagtggggcaagaatatcgtctgcgtggggaggaactatgcagatcacgtcaaagagatgcgcagcaccgtgctgagtgagcctgtgcttttcctgaagccgtccaccgcgtacgctcccgagggctcaccggtgttaatgcccgcttactgccgcaacctccaccacgaggtggagttgggagtgcttctgggcaagcgtggtgaagcgatcccggaggctgcagccatggactacgtggccggctatgccctgtgcctggacatgactgccagagatgtgcaggaggagtgcaagaagaaggggctgccgtggaccctggctaagagctttacgtcctcctgcccggtcagtgccttcgtgcccaaggagaagattcctgaccctcatgccctaagactgtggctcaaggtcaacggagagctcaggcaggagggcaaaacatcatctatgatcttttccatcccctacatcatcagctatgtttctaagataataaccttggaagaaggagatcttatcttgaccgggactccaaagggagttgggccaattaaagaaaacgatgagatcgaggccggcatagatggggtggttagtatgaggttcaaggtgaaaagatcagaatactgagagtagagtgccaaaggggaagggagacagaagcaagggaaataaatgacactaataatgaaaatctaaaaattatgctagacatgtcaaaaaagatgaatccttaaaaaataacgtgatctaaaagacctgggactgaaatagaaacaggaacaacgaagctaaaggatatagaatgcttcccaagagggcaatgttaaactaaactttgagaagttgtagttttcttttctaaaactgaactgaatagacttttcaataaatcattctgaagtctatacctccagacttaaaagtgcaaaggaagtaactcattaatgctgttggcctttaggaaagctgtttgtacagagcactccagttgatttttacttgggaaagtcctccacctcaaagccagataagaattttgttaagtgagctcttgctgcagccctgccttgtttactttgggtgtggcttctgagtttggaagataattttgaaattgttttccaaataaagaccatttctgtgtgactgaaaagagcactttcttttcccagggtggggagcacttcttcctctgggacttaggaccttggcctttggtttcctgagcactgtgcctctttaacttattaaccacataaaccaatacttccacctcaaagacacaggccctagggaactgacaagcctggagatgctgtgtgtgttcatgcttgcacccctccctgataaaatgcctgtcaggatgcacagcacagttgtcagggcctagtggagtcttgtagaattagatagtggcttttgctgagagggtacataggagcctgctggtccctttaagaaggcatgttacatcttgttagtagtttatcattactgtaaggcaatatgccaaccatagttcattccatatttctgtgtattctgggtttctTGTTTGTTTTTTTTGTTTGTTTTGCAAAGCTATACCACTGGCATTATTATTATTTCTGATGACAATGCAGACTTAGAAGATAGAACTGAAGTAGAGCCAAGTTGTAATGAAATTGTCACAAACTCAGAACCTGAAGTTTTAACTACAGCATAGGTAGTGTCATATAAGTAGATATATAAGAAAATTTAAGCATTTAGGTATTCTGTGAGCCCCATGCTTAATTATTTATTATGAGGTCCCATAGTGCTAT
This mutation is a 1932 bp deletion beginning at Chromosome 17 position 24,848,493 bp and ending after 24,850,424 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 50866 | GCT AGG GCA GCT AGG GAA CA | Wild type Reverse | A | |||
| 52524 | CAC GCC CTC AAT CCA TCA G | Common | A | |||
| 52525 | CTT CTA AGT CTG CAT TGT CAT CAG | Mutant Reverse | A | |||
| 52526 | Fluorophore-1 | ACC AGT GCC TGT CTC GCA C | Quencher-1 | WT Probe | ||
| 52527 | Fluorophore-2 | TGC AAA GCT ATA CCA CTG GCA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 50866 | 0.40 uM |
| 52524 | 0.40 uM |
| 52525 | 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.