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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= 74 bp
Wild Type = 86 bp
Sequence
wt sequence with deletion in lower case
ATGACAACAACAAACCTGTGTTTTCCCTACTTGCTGTGATAAAGGCAGAGAACAGTATCTGCCTGCACCTACAAATACACTTTCCTGTATCATCCAAAGCCATTTAGAAAACCACATGAGAACATTTCTAACACGCTTTCCTTTATAACACAATGCTGCAATAGCTTACCCATTtacagcactgtagagatcatttcatagttaatcccattaatactgaaaataatatctaatagtgcttctaagttggaagtcatatttcagacacttactaggaaaaatgagtagtgcatcagaactgctagtcactgctgttgtccagagcagtgtctggacaaaatagaactcagtaaatgcttgttgaatgaatcaatgggcttttcttttcaggcctgtattgatgaaaatttggacatggtgaagtttctggtggagaacagagccaatgtaaaccagcaggacaacgagggctggacacccctgcatgcagcagcttcctgtggttatctcaacatagcagagtgagtgagcggcgttgtgggtttgatgcacattcctcctctgcttcccagaaatggaatttcacaaatagttataaatttctctaaattacaaaagcagaaaattgactattaagagctagctgacactgtgctttgtatctcaatgcacagttcttagctccaatttttgtttgcttctgtttttgtatagttcataatttctggactattcagaggcagcatcagcctggcATGCTGTCTGGCTACTTTCCAAACAGCAGAACACAAGCATGGGAGTTGTAGTTTGTTTGTTTGTTTTTTTAACTTTTCTGTAAACCTGCTTTTTGTTTGTTTGTCTTTATAAATATTATTGCAGTAAATAGCAAATTCTTAGCTGGAACAGATGGAACCAAGGCAAAG
This mutation is a 581 bp deletion beginning at Chromosome 1 position 134,902,144 bp and ending after 134,902,724 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44171 | GGT GGA GAA CAG AGC CAA TG | Wild type Forward | A | |||
| 44172 | TGT TGA GAT AAC CAC AGG AAG C | Wild type Reverse | A | |||
| 44173 | CAA CTC CCA TGC TTG TGT TC | Mutant Reverse | A | |||
| 44174 | CAC AAT GCT GCA ATA GCT TAC C | Mutant Forward | A | |||
| 44175 | Fluorophore-1 | ACA ACG AGG GCT GGA CAC C | Quencher-1 | WT Probe | ||
| 44176 | Fluorophore-2 | CTG TCT GGC TAC TTT CCA AAC AGC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44171 | 0.40 uM |
| 44172 | 0.40 uM |
| 44173 | 0.40 uM |
| 44174 | 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.