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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= 85 bp
Wild Type = 88 bp
Sequence
Genomic sequence with deletion in lower case
AAGCAATCTAGAGCTTAAGCCTAGGGTGGCAGATGTGTGGAAGAACACAGACACAGTTGTGCAGAGCCTGGGAAACGGCTTGGGcttccagggaagaggtttatgttatcgttgtttgggttgggttgtttatttctgggggctgggggagggaaggtatgtatgttttgttgtttagtatctcatgtagccaggatggccttgaactcactatgtagctcagactgacgtggaattccaggttctctctttactccccacactggtagctgtgcaccataaaacctggcttatactttgtaaaatcccaatattctcttgcttgctttcagcacccttatcacatgtgtggattctgggatcctcagagtctggtgtgaaaatgacaaagaggcgtcctctgacccagtaaggtttacctgtcccggtggttgaaaggatgggagctagggtaggggtctgatggggaatttggtaaaggaaagattaagggctttgagactaactcatctctggttattttcagctcttggaactgaaggtgggtcctggggtgtgtaggatgcgccaagatcccacacatacacatgtggttgccacatgtgggaaagaaaatgctttgaaagtatgggacctacaggggtctgaggagcctgtattcagggccaaaaatgtgagtgagtgatgaagggttggggagatgctatgtttgattgaggttggggaagtgaaagccactgacatgacctagatggctttgggggtatggaggattggaggaaacaggtcaggcttatcaaactttgtgcccatatgtaccttccacaccaggtgcggaatgattggcttgatctccgagttcctatttgggaccaagatacacagttcctccctgggtcacagaaacttgtcacatgcacagggtaccaccaggtgaggctctttttactcccggattgaagtagcttgaattcagaaaaacaacaaaaaaagtgtctcctgatagagtaggtaggttgcatggtgtaactctgcctgggttatcaaaggagctcatactttcttagtcaacaaagCCCATGGCTGAGCCTACTACTTTCCCCTTACTGCATTCCAGGGTGGAATTCTGAACTCTGGGTTTCTATTTCTCCTTCCTAGAAG
This mutation is a 992 bp deletion beginning at Chromosome 19 position 8,737,406 bp and ending after 8,738,397 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44407 | TGT GTG GAA GAA CAC AGA CAC A | Mutant Forward | A | |||
| 44408 | GCA GTA AGG GGA AAG TAG TAG GC | Mutant Reverse | A | |||
| 44409 | CGT CCT CTG ACC CAG TAA GG | Wild type Forward | A | |||
| 44410 | TCC TTT ACC AAA TTC CCC ATC | Wild type Reverse | A | |||
| 44411 | Fluorophore-1 | AGC CTG GGA AAC GGC TTG G | Quencher-1 | MUT Probe | ||
| 44412 | Fluorophore-2 | AGG ATG GGA GCT AGG GTA GG | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44407 | 0.40 uM |
| 44408 | 0.40 uM |
| 44409 | 0.40 uM |
| 44410 | 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.