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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
>chr6:136401853-136401985 133bp GCCTGGCTAAGAGACAACCTC ACTAGTGTGGAAGCGAAGTGG
Mut = 149 bp
Wt = 133 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
ATGAGAAAAATTAATTTCTTCTCATGGAGAAAATGAAGTCTCTAGCCTGGGTGGAGATCCCACATCAGTTGGGCTGGCCTATTGCCTGGCTAAGAGACAACCTCTTGGGAAGATGgattctccatgctaaaggggacagcaagaatatttattcctttctaagataatacatttttgatcaggcgtcttgccgggccacttcgcttccacactagtctcggaggagtggagaggaaggatgtcgtcaggtcttaaggctgagctggagttcctagcatccatcttccacaaggaccacgagcggctgcgcatcgtgagctggcacctggatgacctacagtgtcagttcctggtgccggaggcaactgtgggcagtccgccctcgccgccaccgctcacccttcactgcaccatcaccgagtcctacccatcttcaccggcgatgtggttcgtggactctgacgatccagatctcacatcgatcctggaacgcctggaaaattcgaagccagacagttcgcttagtcagcagctcaagtggctgatctgtgaactctgccgcttatataacctgcctgagcacctggatgtggagatgctggaccagccggtccccatcgctcagaaggccaccatcaaagatgggtcttcagaagaagacgaagatgcatcttacggactggacgactcggattattatgacatgaaagaagaagagacgagtagtgagaaaacgtcagagaatgaagggcttgagaaagaggatttggaaatattagagaatatccagaaaaacctttggcaaaagaatgcaagtggtgccacgtctggctcagtgtgtgcttcagatagacttatgaaagaactcagagaaatatacagatcgcagagttacaaatcagggactttttcagtggaactgataaatgacagcttatatgactggcacgttaaactgaggaaggttgaccctgatagttgcttgtacagagaccttcagcgcttgaaacaaaaagaaggcatagactatattttgcttaacttctctttcaaagataactttccgtttgatcctccatttgttcgagtcgtgttgcctgtactctctgatggttatgtcttggatgggggggcattgtgtatggaactgcttaccaatcagggctggagcagcgcctactccatagaatcggtcatcttgcagatcaatgccaccttagtcaaaggcaaggccagggtgcgctttggagtagataatcactatactgaacaagtggctagacgggtctataaatccatggtgctgaagcatgagaaatcaggctggttcactcctccacagaaagatgggtaaatagtagagcatcatgtatttggtatttgtagcagtagtgagaaagttattagctaattggtgctttagtggataatctctcaactgaccaccagtagaagtgtcagcctttccattttgcttactgtgggggtcttgggctgaactgtgagccttactgtggtttttcctgataaataatcataactccattttcagtcttccatgttccttgattaagtgaaatgtgttaagtaggccagtcagttgtgactttagataatgctggttattaggctactgggggggggataatgagtatttttaaacatttgttccatttagtactaataaaagtttttccagtgtccaatactgttttagaaaataaaataaaataaaataaaaaaagaataaaaaagaatacatttttttcttcccagaattcctgcccttgtgCAGTTGCTATGGTGTCAACTGTTAGGCCTGTCCATCTTTTTAAATCCTGACTCAGACAGGTACAACTTTATCTTTAAAACACAAAACAAAAAAATAACAAAACCCAACCAACTCCTTTTTTCTTTCTTTCTTTTTTTTTTAATCTGACAGACAGCAACTTTAT
This mutation is a 1677 bp deletion beginning at Chromosome 6 position 136,400,277 bp and ending after 136,401,953 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 50611 | GCC TGG CTA AGA GAC AAC CTC | Common | A | |||
| 50612 | ACT AGT GTG GAA GCG AAG TGG | Wild type Reverse | A | |||
| 50613 | AAG GAG TTG GTT GGG TTT TG | Mutant Reverse | A | |||
| 50614 | Fluorophore-1 | CAT GCT AAA GGG GAC AGC AAG | Quencher-1 | WT Probe | ||
| 50615 | Fluorophore-2 | AGA TGC AGT TGC TAT GGT GTC AAC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 50611 | 0.40 uM |
| 50612 | 0.40 uM |
| 50613 | 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.