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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
>chr18:37400953+37401101 149bp TTATGAGCAACACGGTGAAGA CATTACTTGCCTGTTTAGGTGA
Mut = 140 bp
Wt = 149 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
TTTGACACTAAAGGGTTGTTTGCCTTAAAGCTAGAATTGTATCAATTATCCCAAAAAGACACAGGGTGGCGCTGCAGGCTAAGATTGTGATTAAAAAATCACTAAAATACCCGGAATCACTGTTGCAATACAGAACAAAGGAAGCAGACAAGAGCTTTGCAAGCACTATGGAGCTATAGAGGGTTTGGGGCAAGAATATAATGGATTAATATGAGATTATGAGCAACACGGTGAAGATTCCTCCAAAgagggaatcagaattcagtgtttcaaaacatttggagactgctacaggattggatgcaagcatgcatttcctcatcatggagaagctggggagaattcacctaaacaggcaagtaatggcatttatttttatgatggttttggttcaggtttgcagtgagccaacaattcgatactctatcttagaggaaacagaaagtggctcctttgtagcccacctggccaaggatctgggtctgggagctagggagctggctgccaggtctgcacgggtgttgtctgatgattacaagcagagattactgctggatcctgagactggagatttgcttctgagggagaaagtagaccgggaggaggtgtgtagcactgtggatccctgtgtgctgcatttccaggtgactcttgaaaagccagtgcaatattttcaaagagaattattgatccaggacataaatgatcatgctccagaattcccagatagagaactgctcttgagaataccagagaacagccagcaaggcacacaattttcactgaatttagcccaggacttggatgtgggcagcaatgggcttcaacaatacacagtcagccccaacccttattttcacgtcctcactcaaaataatagtaaaggcaaaaaatacccagagttggtgcaggacagaggcctggacagagaggagcaggcagagctgagcttaaccctcacagctctggatggcgggtctccaccgaggtcaggaacagccctggttcgaatcctgatcatggatatcaatgacaatgctcctgagtttgtgaacagtccctatgaggtgcaggtcctagagagcagtcctccagactccccagtcttgactgtcttagcacgggatgcagatgctggcaactttgggagagtttcctatggcttctttcaagcatcagatgaaatccaacaaacattctcaataaacgcaactagtggagacatgcgattgaaaaagaaattggattttgaaaaaattaaatcttaccatgtggaaatcgaggccatagatggaggtgggctttctgggaaaggctctgtaacgatagaggtggtggatgtgaatgacaatgccccagagctgaccatatcttcactgaccagctctgtcccagaaaatgctcctgagacgatcataagcatcttcagagttggggacagggattccggagagaatggaaagatggtttgttctattccagagaatctgccatttattctgaaatccaccttcaagaacttctacaccctggtgacagagagccctctggacagagaaagcagagctgagtacaatatcaccatcatggtctctgacatgggcacacccaggctcacaacatggcacaccataaaggtgcaggtgtccgacatcaacgacaacacccccgccttcacccaaacctcctacaccatgtttgtccgcgagaacaacagccctgccctgcacataggcaccatcagtgccacagactcagactcaggctccaatgcccacatcacctactcgctgctgccgccccatgatccggaactggccctctcttcgttaatctcgattaacgctgacaatgggcagctgtttgcactcagggcgctggactatgaggccctgcaggtcttcgagttccacgtgggcgccacagatggaggctcacctccacttagcagccaggctctggtgcgcgtggtggtgctggatgacaatgacaatgcgcccttcgtgctctaccctatgcagaacgcctcggctcccttcactgagctgctgcctagagctgcggagccaggctacctggtcaccaaggtggttgcagtggaccgcgactctggccagaatgcctggctatccttccagttgctcaaggccacggaacctgggctgttcagcgtgtgggcgcacaatggagaggtgcgcaccaccaggttgttgagtgagcgcgatgtgcccaagcacaggctactgctggtggtcaaggacaatggagagcctcagcgctctgccagtgtcactctgcaggtgctgttagtggatggcttctcacaatcttacttacctcttccagaggtagcgcgggatcccgcccatgaagatgaggacgtgcttacattgtacctggtcattgccttggcatctgtgtcttctctcttcctcctgtctgtgctgctgtttgtgggggtaagactgtgcagaagggccagagaggtctctctgggtggctgctctatgcctggtgaacactttcctggccacctggtcgatgtcagtggagcagggaccctgtcccaaagctaccagtatgaggtgtgtctgaggggcgactctgggactggtgagttcaagttcctgaaacccatgatccccaatgctgggatagaaattatggaaagtccccactGCAGGGATAGCTTTGTATTCAACTAAGTAATGAATCTTAAGAATTAATACACTTTTATATGGTTAAGTGTAACATTAACTAACTTTAGGAGAAACTACCTTGTGCATGTCTCCAACCGCTTTTGTTTATCTTATTCATATTACGCACGCTTAGTCT
This mutation is a 2432 bp deletion beginning at Chromosome 18 position 37,400,984 bp and ending after 37,403,415 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48946 | GAC ATG CAC AAG GTA GTT TCT CC | Mutant Reverse | A | |||
| 48947 | Fluorophore-1 | ACA TTT GGA GAC TGC TAC AGG ATT | Quencher-1 | WT Probe | ||
| 50580 | TTA TGA GCA ACA CGG TGA AGA | Common | A | |||
| 50581 | CAT TAC TTG CCT GTT TAG GTG A | Wild type Reverse | A | |||
| 50582 | Fluorophore-2 | TCC AAA GCA GGG ATA GCT TTG TA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48946 | 0.40 uM |
| 50580 | 0.40 uM |
| 50581 | 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.