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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
>chr2:18715442+18715541 100bp GCATTTTCAGCACAGTTTGA TGGGAACTGCCTGGGATATG
Mut= 107 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TTAATATGATTTTTAGGTATATATCAGAGCATAGCTGCTATTCAGTGTAGTTCACTGAGGTCAGGAAAGTCTGCCATACTGAGAGGCTGAGGGGAAATAACAGAAAAGAGGTGGTCAGTCAACAGAGTTGGTTAATAGGCTAACATTCCAGAGTTCCCCTGAGTCAAACAGTACCTTTTAGTATCAAGCAGATGTAGAGAATCCAATTATTATATGAATTTGCAGAAAGTCCCATGTTTATATCTGATTTTATATAATTTCCAAATACATGCTTAAAAGTAAAGCATTTTCAGCACAGTTTGAAACTGACCTTGCTTCTAGGTTAGTTACTAACcagcggcaatgggagcgataaagactcaacatatcccaggcagttcccaccgtctgcgttccgaggcggttgtgctttccttcaatgccagttttgtgtcactttcaggctattttagattctaggtttggtctctttttccctgtagtgtgtctacaagaaggcagctgcgtttgtgctgcgagcggttggtaaacactctcctcagctggcgcaggccacagttgactgtggtgccctggactcactggtgatctgcttggaagattttgaccctggagtcaaggaggctgcagcctgggcacttgcatatattgcaaggcataatgcaggtactggggtgtgcttgaagatagactcaagtttaactttactagataagaacagttttgtttattatccattgatggagttttaaaagttgctggacattatgggagagctgtcttgaaggatagccacactgatttggatcacataattacaaaacacttctacatttgagttctagcagaagggaatctgaaggagacGGTGGTATGGTAGAGTGGGATTGGCATCCAGTAGCAAAGGTACATGCCTATAATCCCAGTATTCAAGAAGCAGACTCAGAAAGCTTAAAGTGAGCTCTGCAAGCCTGAGTTACACAGCCAGACACTGTCACAAAACCAAAACAACTAAGTCAGATGAAGAAAGTTCAGCCGACCAGGTAAATATATTCTTACTAACTTGTCAGAAGACTATTACTGGTTTCTCATGCTCTGAGCTCCTTGTCATTCACGACAGGACATTTCAAGCCAATCAAGGAGCCAGATCACATGGATGAATGGTGCGGTAAGATAATATACAGTTGTAGAAAATGTTTTATTACCCTGTGTTAGCAGGAAGGAGGAGGAAGAAAATAAGGGCAACACTTTTCTCAAAACTGAAATCCTGGTGTGGTCAACAAGACAAGACGGGGCTCTCCTGAGCGAAGACT
This mutation is a 533 bp deletion beginning at Chromosome 2 position 18,715,493 bp and ending after 18,716,025 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48272 | GCA TTT TCA GCA CAG TTT GA | Common | A | |||
| 48273 | TGG GAA CTG CCT GGG ATA TG | Wild type Reverse | A | |||
| 48274 | GGA TTA TAG GCA TGT ACC TTT GC | Mutant Reverse | A | |||
| 48275 | Fluorophore-1 | AAT GGG AGC GAT AAA GAC TCA A | Quencher-1 | WT Probe | ||
| 48276 | Fluorophore-2 | ACT AAC GGT GGT ATG GTA GAG TGG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48272 | 0.40 uM |
| 48273 | 0.40 uM |
| 48274 | 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.