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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
>chr8:123845651-123845767 117bp AGCTCCTGCTACGGAAACG CCAGCTATGGAAACAAATTACCAG
Mut= 123 bp
Wt= 117 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GAGTGACTTAGAAGCCAGTCTTCAGCAGCTGCCCAGTAAGGACAGCCTAGTCTGTCCGCAAAGGAAGCATCCAATAGAGAAGCCCTCAGGGGCCTGTGGAGGGAGCTCCTGCTACGGAAACGCTGCTCATTTGGATAGCGATGGTGActcctgtggatgggcatcacgggtgctttggaatagttttggtttaatgctggtaatttgtttccatagctggtttaagtaaaacctgtagagttaaacatgtgactgacttgatgaaacattatttttcaacttaagtcatatatatgtatatatgtatgtgtgtgtgtgtatatatatatatatatatatatatatgtatgtatgtgtatatatatatatgaacttgaactttaaagtttttccatctcatcttgtcccttttttttttccaaaagcaccaccaaaaaaggacacggaagagaaacccgaagaacacaagacaaaagagactgatggggcacccagtggccctggaccgcggcaacagcctagtgccttatgtgccagaggaagtaagaaagccaccagaagcccacagagatcgacgagcaagataaaagagaacaagcacccgtttgctctttatggctggggagagagacagatggacatgggaagccagaagacacacaacgtctgcgcgtcagcctctgtgcacgaggtgtgccttgaacggggccagactagccctgagctgccttgggggcatttctcctatggctacggtgcccagcgccactggaagtccttgggttatgtgggagtccatggaagccaaaagaaggggtctctggtcctagaaagatggagtggactttggttaaCTGGGGCTACAGCCAGGGCTAAGGTCTGATGACAGGAAGAGGTAGCTGTCATTGTGGTGTATGAGTTCTGTGGTCTGGGCCACAGGGATAATGGGAGCAGGAGTTATCCCCTGGGGAGGCATC
This mutation is a 707 bp deletion beginning at Chromosome 8 position 123,845,017 bp and ending after 123,845,723 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 43583 | AGC TCC TGC TAC GGA AAC G | Common | A | |||
| 43584 | CCA GCT ATG GAA ACA AAT TAC CAG | Wild type Reverse | A | |||
| 43585 | CCC AGA CCA CAG AAC TCA TAC A | Mutant Reverse | A | |||
| 43586 | Fluorophore-1 | CCT GTG GAT GGG CAT CAC | Quencher-1 | WT Probe | ||
| 43587 | Fluorophore-2 | CTG GGG CTA CAG CCA GG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 43583 | 0.40 uM |
| 43584 | 0.40 uM |
| 43585 | 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.