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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
>chr5:53276821+53276927 107bp TTGTGCCTGTCCCTTCTTTG TCTCAGCATTTTGGAAAGGAC
Mut= 103 bp
Wt= 107 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (TG insertion):
ATGCTTGATTTCTCTTGGCCTCTGCCTGTTGTGCATCTCCAAAGTGCCAGGCTTAGCTGCTGCAGCTACAGCAAGCTCACTAATTGGCACAGCACCTGCAATCAATTAGGGAAGCTTCAATTATCCAAACCAAAGCCACCCACCCAAAGATTAATTTTCTGCTCTATTTCCTATCAAAAACATAGCATATTTAGCTTGGAGTCCAGTCCTTGAGTGGAGTGTTTTTAGCCACCGTTTTTCTGATAATTGTGCCTGTCCCTTCTTTGCGGCAGCTCTCCTCT^tgtaatgtcacacccaccagctctttgaaacagttctttctgttgtgttgtgtcctttccaaaatgctgagatcacaggaaaaagatgttggttgggatctttttgtttgaagtgtggctctttttacatctaggatctagcttctataacctaaaagctttaagtaatgacgtccctgaattgaatcacactcctaaatatgcttgtaattagcccatataaatagaagcaaaatgcccacatctcctttctaaagagtaattatttgttctgttgtgtttctcagagaaggagcaggctgtaaatcgagctggtattgtccaggaagatgtgcaaccgccaggtaaactgaaaacgctcagacaatcccttccggggcacgtggatgagtactttgctgtggagtgggtggttcttctccctggaacctgatactgttcattattttatgatagttcctctcttgtcagggctatctaagacttcatgctaatatttcagtgcctagtactttttacaagcttcctgggcccctcgtttgtccttggtaagtggacataaaatggaatcaagtatgcaacagatcatctgctttgcagactcccatactgagcacccctgtaaatacctccctcactggcagaccgtgtgtgagggtgagcactaaggttcagtttcctagagaagttctcaaagctcccttcagggacgtgtgggatgttcctttaaaatgatacaagttggctgagtacgagagattttctagcttattaaccaaactcctgcagtctggtggtcctaggctagatccatttcttcctagcagggaagagttcctaagcctttaaaaggatacctcatagctgccatggctgcctcgttgcagggttccagtggctgcactcacccatttcaatgtctcagccacatggaaattgcatggctcaatttccacatcatcttgtttgtttgggatcaagggggtgtgttcagtcatttagcaatgaatcctgatagggtatttgtgtttctttccatagggttgaaagtgtggtctgatccatttggcaggaaatgaggctgtcagcaagtctgatgaggaaagtggacgtctttatcctgtgcactccgcagtggggacaatagatgcctcactgtggcagcatggcatggagagggaactctcatgctgctagccagaccccttgtgatagagactgtgtgcaaagacagtgcttcccttaactccctggagaacctgaacagatgccaccattaggaagtgccttgcggctccattgactttgcaggagcagagccagcctgcaaggctgtttgtggaagatctgctgctcctgcagtctttatcacttccaagctgtgatgtgaacacaagcaacctgtgggctcaaggtccgtggctgctctgacaccttctgaataagcgatttcagtgcaaatggccttgccaagctgcctcgcagggctcttggaggatgtttcagttgataaaactgtttgaagacaggatccttggcactgtttaagaatatacactgctcagcttaaccatctcattgaaagtcactgtgtgtggaagtgaatagggagcgagtcacactagactataccacacacagtagactcctgcgtgaggctgcaggtattaaaatggtttctcttagtttgctggtgtgtactgtttttatattttgtgtgtgtatatgtttgttttatttggatgggtggtgtttgttggctttggattttgttttttgagacagggtctgtcagaccagtcaagccttgaactcccagagatccacctgtcactgcccagtagaatttttttgtaaattgaattctgatgtgaagaagtagaagtagtgctattgtctagattctatttgagtagatatccgtgtagt^AATAGCTATTTGTAGACAAGGTGAGCGGCACACTTCTTGTGGCAGCTGTGGCATTAGGTTCTCCAGAGAAACAGAACTGATGAGATGAATATGATTTACATCAGTGACTTACAGGCTGTGGTTCACCTAATCCAACCATGCCTGTCTTATCAATGGAAAGTCCAAGAATCCAATAGTTGTTCAGTCCAAAAGGCTGGACTTTAGTATCTACCAGAATCCCGAAAAAGTCGTCTGTAATCCCCGTGAGAGCAACGGCAAGCAGGCAGAGAGCACACA
This mutation is a 1929 bp deletion beginning at Chromosome 5 position 53,276,856 bp and ending after 53,278,784 bp (GRCm38/mm10). In addition, there is a 2 bp TG insertion at the deletion site.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48494 | TTG TGC CTG TCC CTT CTT TG | Common | A | |||
| 48495 | TCT CAG CAT TTT GGA AAG GAC | Wild type Reverse | A | |||
| 48496 | CTG GAG AAC CTA ATG CCA CAG | Mutant Reverse | A | |||
| 48497 | Fluorophore-1 | AAT GTC ACA CCC ACC AGC TC | Quencher-1 | WT Probe | ||
| 48498 | Fluorophore-2 | ACA AGG TGA GCG GCA CAC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48494 | 0.40 uM |
| 48495 | 0.40 uM |
| 48496 | 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.