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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
Mutant= 74 bp
Wild Type = 88 bp
Sequence
Wt sequence with deletion in lowercase
GGTTGGAATCTGAGTAGTGCATAACAGCATTAGGGTACATGCCTGAGAGGGACAGAGGAATCAGCTTCAAGGTCATTCCTGTATAAGCATCAAGTTTGAGAGCAACTTGGGACACACACACAGGTCCCTATCTCAGAGAGGAAAAAAAATCGCAAGCTTGCCTGTGGCCGAGGACGGGCGTGCATGTGTGCAGTTTCCTCTCTGAACCTTAGGTTTTGGTCCAGAagttggaagtgtgggaagaacggggaaaattgagggaaagggaccgaccaggcaactcctggaggcggtggggtgtgaggggctgtggctgccccgtctctcacttgtgcctctgtccctgcaggtgagtcagtggccagctctatgcattcctcgcgctaccctagcccagcggagctggatgcctacgcggagaaggtagccaacagtcccctgtccatcaagatcttccccaccaacatccgagtgccgcagcacaagcacctgagccgcacggtcaatggctacgacaccagcggccagcgttacagcccctaccctcagcacaccgccggctaccagggccttctggccatcgtcaaggccgccgtctcctcgagtgctcatgcgcccgccggccaccccaaaagcgtgctcaagagtgtggagggcaagcggactaaactgtcgccggccaccgtgcaggtgggcattgcgccttacccagtgcccagcaccctggggcccttggcctacccgaagccacccgaggcacctgcaccaccgcccagcctgcctgcagcagccactgccacctctgtgatccctctgcccggccggggccttcccctccccccctccaacctaccctctatccatagcatcctgtaccagctcaaccagcaatgccaggccccgggtgctgcaccaagcgcctgtcagggcgtggctgtgccccaccccagtccagctaagcatggcccagtccccagctttcccaacctggcttactcggccacagccggtctgccagactgccggaagggcacagaactgagccagggagccaccccagccctgacactggctggggccaccaaacctgcagggtatgcagaaggtggcctggattacctgctgtggccacagaagccacccccacccccaccccagccgctgcgggcctacagtagcagcaccgtagccggcaagtcccagtccccagagatttgtggcgggagggccttcgaaagggccaacgggtcgccccacaactgcggaatggggctgcccggcagcttcaccgtgggccagtacttcgctgccccttggaacagtgttctggtgacacctaccagtgactgctacaatccagcagctgctgccgtagtgacagagctggcaccaggagccgcccgggagctagccgggcctcctggagacgtactctcgggcctgaccagcaagagtgtgtgtaacacagcggtgctgagcagcagcctccagtcgctggagtatctcatcaacgacatccggccaccctgcatcaaagaacagatgctgggtaagggttacgagacggtggccgtgccccggctgctggaccaccagcacgcccacatccgcctacctgtctacagataaggtctgccctgcggacacacggacagggtgatgggggttgccaggccatagcacagggcgggaagctagcagggcgccccgcccgccctgtgtccacggggaagccaggctggttacctcctcacttcagtgggcaggcgggttgacctcacacaccagggcttctttcctctcccgcttgctgccccaggcagcagggagggtcccaggctggccacgaggctccttcatctttcatccaggcagggaaagaggaccttctgccaacaagaagttccttctgggtctccagcagagggcgctccagtcatcccgtgtcatacacggggtctctatccacaatctcccagtccctgggtccaaagtgccccaccccaccaactaatccaggtgaagaccgccctgactccacacccccagaggtctcaggactaccccccccccgttcctgctgactctccccctacccacccaccctccctactaggaatcacgcccaacctacccggtacgccccagaccaacctcttctccatccctacactacatctcacagggctgctgggaacagttggtgcgttgcacaagggcaccttgcccaaggagactccacacacatcctagatttgtgtatttattacagttgtctgcatttcaaggaagggacactatcttcctattcgcacaaagatgtcacggggggctaacaagggacctgtaaattcaggctccattcacaaggacggtcttgccttcttcgcatctctgggtctcctggggaccagtgggagggtgacctggctgtaactggaatacccaatccagctcccgttctcattccccccacccccaccgcactgctgcggggcgtggggaagagagaagcccaggaagtcctcaaggcctgcagtccccgggccctgccggccccaggcgctccggaaagcggtactgtatctcgccaaggcctgtctgagcactaagtgcatttacaaagctctgagaatgttttttttatactaaaattgaccattatattctactgtgagaagtgcagtctgtctgcactatattgttttaaaaacgaagagaaagaaaaaaaacggaaaacagatggtgtctcagctgtgggtttgctgtaaggctgttctctctctctccctggcttcccccacctcagggatccacgggagggcaccccaaggagccctgtggggacagagtggggtggcatgcagggtgagctatgcccaacttattacaaagctcttcaagataaatgctatcccgtgtctatgtgtcaggcccgtggTCCATCCATAGGGGCAAAATCAATCCTGAGCCCAAGCCCTGGTGGCTCTTTTCGGTGCTGAGGTGGCTCTGAAAACTCCCCATTGGAAGTCCATCTCTAGATCCTGTTGTCATCCTCCCAGATTAAATGGTTGCCATGTAGCTGAGAACAGGCTAGCCATCGCCCTGGCCGGCATGGATTATGCAGGCTGACACCTGGCATATACCCATACCC
This mutation is a 2716 bp deletion beginning at Chromosome 5 position 114,610,703 bp and ending after 114,613,418 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44244 | TGC AGT TTC CTC TCT GAA CCT | Mutant Forward | A | |||
| 44245 | GCT TGG GCT CAG GAT TGA | Mutant Reverse | A | |||
| 44246 | CCA CCA ACT AAT CCA GGT GA | Wild type Forward | A | |||
| 44247 | GGG AGA GTC AGC AGG AAC G | Wild type Reverse | A | |||
| 44248 | Fluorophore-1 | CCA GAT CCA TCC ATA GGG GC | Quencher-1 | MUT Probe | ||
| 44249 | Fluorophore-2 | CCG CCC TGA CTC CAC ACC | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44244 | 0.40 uM |
| 44245 | 0.40 uM |
| 44246 | 0.40 uM |
| 44247 | 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.