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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
>chr18:76941148+76941268 121bp TTGACCTGAACAGTGAGAGCA TATGGGCTGATTTTGCCAAC
Mut= 125 bp
Wt= 121 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
AATGCACTTTAAAATTTGCCCACTCAGGCGTTTTCATACTGATACTTGGTTAGATGACAGAACTCTTAGACTGAATTCGTAGTTACTCACCCTAGCACTGCATTTGTTTTTTAGTTCTTCATTGACAAAAGATTCTTTTGATAATTTTGAAAAAGGAAAAAAAAAATCAGGTTGTGACAGTTGCcatctttactgaactgaacaccatgaaatcatttgaactgtagattttgtaaaatcatagtgaaatgaagaaaatatggttgttgccactatcaaaagaaaaaaattaaagaactcaatattttaactttaagatgtatgagtttatacttaatatcttcacagggctcaaactagtttttaaaagatggctaatgagggaacaagacaagtgcataatacatttaactcaactaatcatctttgtcttgcacagttggctggggaacagaccagggaatcggtggatttggagaggagccaggaattaaatctcaactaatgaaccttattcggtctgtaagaaccgtgatgagaggtaagaagaacttagaggacgttataagttacagtccgtggtccagtttcagaccccaaagcgtgggtgaatgtgagctctcaggttctctttgtgtggaacatggacttctgtaacctgaatttactgcttcagagttgctggccgctttcttccattgcatctgacttctaggaagttcagtgttactagtacactctgtgtgtttcctgtcagggcagaaagacaaaatgatgtccttactgcttaggaactgtagctgccatcttgtaatgtagagtcatgaagaagtttccaagagtgaaataaaggctctgtattgtattgaaaaatactttggagcaactcttcaatcaagaaagagttcagataccttgtatttaaacaatgggattagagccttttcaaaccttaatcatagctttgggtttattgttaataattttattctttttgatgaaactgcagaaccttccttgaactgtatttctgctccagtgtgcagctgtgtggatctgtctgatcctttgtgttcccagagctgcagcagcaccatccagtggtgcctccaaacacccagacatatggtggggtggggcatggtggtccacacctgcagtcccatcccttgggagggctacagaggcattcctggttcacctccatccaacaaaagaaaatgtctttggttttatggtcatttatttttctgttattactattactatatgattatgatggcatatttatagatacttccatactcctcggaagctatttattgctgtttaattttttgtttgtttgttttttatactaacaaactttatttttttcttttagtgccattgataatagtaaactcaattactattgtattgcttctgttatttggctgaacatcgttgggaaatcagagactgagaagaagaaaagccccagaagttactttctgttgtgaccggaatattcatgtttcagcaggctcatcttgcagttgacaaaaaaaaaaagaaaaaaaaagtaaagttgacaataaaaccagagtttctatctatctgatttttttttttttttaattgtagttgcacttgtagatcagattatcagaggcaggagcactcccggattggagcatacttgttttagctaaaataggtaaaggctaggaaagggcacaaaactgctgcctttgtgtcactccatacgacaccctgttacagctcagggatggatttagatataatctgagagtatataacttcctatataaacttaagagtttttttaaagtaacagttacatctagttgacaaattggtatttgggtaatttattttgaactctttggttacctcgtttctgctataaagaaatttgtaggtaactctacggggcaatgccgtgatttcagagagaattctaattctggaaaccagttgtttgggaactgtggtttttacgatattatttattagttgacctgaacagtgagagcagactgcttacatattttcctttgtaggaagtcttgcctgttttacaaggatttgttttataaaaatcagtgtaactgttagttggcaaaatcagcccatacagtcagactagcctgctcccgtgactgtctgttaggaagcctttgccttccttaggttgtgcttccattcctcggcggagtggcatgctgtaggtatgattcttaattctgtctgtaggtaggttgttgattctaagtagctttcgtttgttcgtctgtcttatgtcttagaccagctttgtggtgttcttggctcatctcataatactgttgttcagtttatatcagaagagttaaactgtgcaaatattgtatgaaatgtttcagttaccataatttaaaggggtgggatggcatctttctgtatagaaaacttttataaataaagttgacttttcatattaagcgtctgatttatgcctttatatagacctggcattatgagtttgttgatccctcttttattcttcactgtaaaattgctgagtagaaatttaatgaatgttaatattcataatctatttgtttcttcagtgaattaactgaattgttgacataaatcaagagaatccttgcgtaacttttaagttactgctaactggggcaggtattgaagatctcctggtaatgtctcaattggagagagtgggtctttccttcagtcttcagggctgtcagcggtacCAAAAAAGTGTCCTTGAATCCTTGGCCCCTCCCCTGTGAGGAGCTTGGTCCCAGGATGTTCCCTTGGCCTCAAAGCTAACAGGCCAATCTTGCTGACTCACACCTCATTTTATTTTATTTTTTTTAATTGTTGAAGATGATTGCATCCAAGTTTGAGCCTCAAATCAGATCTTTAAAA
This mutation is a 2581 bp deletion beginning at Chromosome 18 position 76,939,313 bp and ending after 76,941,893 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 40040 | TTG ACC TGA ACA GTG AGA GCA | Wild type Forward | A | |||
| 40041 | TAT GGG CTG ATT TTG CCA AC | Wild type Reverse | A | |||
| 40042 | TCA CCC TAG CAC TGC ATT TG | Mutant Forward | A | |||
| 40043 | GGC CAA GGA TTC AAG GAC AC | Mutant Reverse | A | |||
| 40044 | Fluorophore-1 | TTT GTA GGA AGT CTT GCC TGT TTT A | Quencher-1 | WT Probe | ||
| 40045 | Fluorophore-2 | AGG TTG TGA CAG TTG CCA AAA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 40040 | 0.40 uM |
| 40041 | 0.40 uM |
| 40042 | 0.40 uM |
| 40043 | 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.