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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
>chr18:37683015+37683094 80bp ACCAGTTTCAGTCTTTACTGTTGG GAGTGTTGACTTGTAGGAAATAGTCTC
Mut= 82 bp
Wt= 80 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CAGAAATTAAAACACTAAATACAGAATTTACTTCAATAGATATGTAAAATGTAAGGATCATAATCCACTGAATACTAAGACTGAAAATCCACTTGCTACTTGGGATATGACAGTCGGGCTTGAGTTTCTAATTGCCATACATGAAGACATTGATGGCATTTGTTTCCAAAACTGGAAAAAAAGAAAAACGTGCGCcttgtgaattgggaaggataatatataatgatgaaaacattcatgaaatgataataagcaaaataaaatatctaaaccaaaggggaaaaatgtgaatacctttgctgtaaattaaagtcaaaacaaaagctttgataaatagctgcacgatacgcagttttacaagacaaccattaggcgacgctgttgaccaaagggaacagcagggttctccgttccgcagggtccagcgagggtttgcttgctttttccagcacccatgagaacatccgtccggttcctgtgccctcttcaagcgccgggcaaatcctgccctcaaatcacaaacatctagggcactagctgtctttttgaaatcccagagaaacctcgaaaagaagctacctggcaagctcagaacggcagagcaaccccagcggctttggaacgatggaaaatcaggtattgcttcacttcctggtgcctttgttctgcggcgccctctcccagcccatccactactccattccggaggagctagccaagggctctctagtggggagtctcgccaaggatctgggactcagtatccaagaattgccagctcgaaaactgagagttagtgcagaggattatttcagcgttagtgcagagagtggggatttgttagtgagcggcaggatagacagagaaaagatttgtgggaggaaatctgagtgtgcactggaatttgaatctgttactgaaaacccgatgaacattttccacgtaacggttgccatccaagacattaacgataatgcaccacatttctttgggaaaagcattgaattggagatctgtgagtcagccttagcaggggcaaaattccctctggactctgcgcgagatgcagatgtgggaagcaactccctgaagatgtacactgtcagccccaatccacacttctccttgtcaacgaaggaaagtcctgatggaagtaaatacccagaactgctgctggaaacccctctggacagggagcatcagagctcccatcacttaatactgaccgccatggatggtggagacccgcccctaagcagcaccacccaggtctggatcaaagtcaccgacgccaatgacaatgctccagtgttcagccaggacacttacaaagttagccttcgtgaaaacatacccccaggaacgttagtgctgcaagtgacagcttctgatcaggatgagagtattcacggagagatcacctatgccttccttaatgccccagcaagtactaaccttgtcttcaatctcaatccaaatactggagctatcacaactaatggtacattggattttgaagaaaagaatagatacacattgggtgtagaagccaaggacggaggggtgcacacggctcactgtaatgttcagatcgaaattcttgatgacaacgacaatgccccagaggtgacattcatgtccttttctaaccaagttccagaggattcagtccttggaacagtaatcgccctcattaaagttcgagacaaagattctggacaaaatggtttggtggcatgccatattcaggaagaacttcccttcaaattacaacccacctccaagaattattacaagttagtgattgacaaggccctaaaccgagaacagaccccagagtacaacgtcactatcacagctactgacagtggcaagccctcgctttcctccaagacatgtgttactgtacacatcacagatatcaacgacaatgcaccggtttttcaccaggcctcctacttggtccacgtggctgagaacaacccacctggagcctctattgctcaagtcagcgcctcagatccggatttgggatccaatggcctcatttcctactccatcatagccagcgacctagagccaagggcactatcgtccttcgtgtccgtgaaccaggacagtggagtggtgttcgcgcagcgcgccttcgaccacgagcagctgcgttccttccagctgacactgcaggcgcgggaccacggatcgcccacactcagtgccaatgtgagcatgcgcgtgttggtgggtgaccgcaacgacaacgcaccacgcgtgctgtatcccacacttgagcccgacggctctgcgctcttcgacatggtgccgcgcgctgccgagcccggatacctggtcaccaaggtggtggctgtggacgcagactccggacacaatgcctggctgtcttaccacgtgctgcaggccagcgatcccgggctcttcagcctgggactgcgcacaggcgaggtgcgcacagcgcgtgccctgggagacagggactcagcgcggcagcgccttctggtcgctgtgcgtgatggtggacagccaccgctctccgctacagccacgctgcacctcatcttcgctgacagcctgcaggaggtcctgccagacctcagcgatgaccctctaccatctgaccctcagagtgagcttcagttttacctggtggtggccttggccctgatctcagtgctcttcctcctggcggtgattctggctattgccctgcgtctgagacactcctccagatcagctgcctggggctgctttcagcctggcctcagcctgaagtctcaacctgtggttcctcccaactacaatgaggggacattaccctattcctataatctctgcgttgcttcacattctacgactatggcagggtttaattttcaccacgtggcaccagaggttgctccccctcaggatctcctgtgcgatgatccttctgtggttgtatgtggcaataatgaagatccccaagtctcggacgactctgctttccaggtaagtttctacagagcaacttaaatgttatttgctgtatttacttctcaatcgtgcacaaacctgtcaattccgaacatgttttcatagtttgggggatatttagtggaagacgtgtcgtaggtgcgtaaagattggctgttggttgctcagtcatcttggaaatgacctgattaccagtttcagtctttactgttggtgtttctgtgtggctagcaaatCTTGCCGGAGACTATTTCCTACAAGTCAACACTCAATAATATTAGTGCCCTTTCTTACTTAATTCAGGAGACTGAATTTTAAAATAATCTTTAATTCTTTATAAGCACATTGATATGTCTTTCTGCCTAAAGTTTATGGTCAATTACCTTTCTTGCTTGAACAACTTTTGTTTGTTTGTTTGTTTGTTTGTTTGTT
This mutation is a 3032 bp deletion beginning at Chromosome 18 position 37,680,029 bp and ending after 37,683,060 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 38898 | ACC AGT TTC AGT CTT TAC TGT TGG | Wild type Forward | A | |||
| 38899 | GAG TGT TGA CTT GTA GGA AAT AGT CTC | Common | A | |||
| 38900 | CAT TGA TGG CAT TTG TTT CC | Mutant Forward | A | |||
| 38901 | Fluorophore-1 | TGT TTC TGT GTG GCT AGC AAA T | Quencher-1 | WT Probe | ||
| 38902 | Fluorophore-2 | AAA ACG TGC GCC TTG C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 38898 | 0.40 uM |
| 38899 | 0.40 uM |
| 38900 | 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.