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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
>chr4:10879170+10879260 91bp CATTCCCCAGTCATTCCAGT GAGTGACAGCACAAGGATGC
Mut= 86 bp
Wt= 91 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GCTTGCTCACAAAGAGTTCATCGTGTACAGAGACCATCAACAGCAGCTTCCTCCACCCCTCAGACTGTTTGTTTGTAATAAGCACTGAGTTTCCACACTGATGTTGTATCATCTGATCCCAGCCTCTGTCATTTCCTCATTCCCCAGTCATTCCAGTCAGGTCAGCCCCAAAGCCCTGCAGgttgtgacatagctccatacctgaaaagcatccttgtgctgtcactctagcagtgggacttaagcttgtggtacatctgggtttttttgtttggttggtttggtttggtttttcaagacaggatttctctgtataatcctagctgtcctggaactcactttgtagaccgtggcctcgaactcagaaatccgcctgcctctgcctcccaagtgctgggactaaaggcgtgcacgccacggctggcttggtgcatctttccttaaggtccagatgaaggttcattattgtgtgtgcatgctttcttttcagatcaacagaaacatttaaaaaagaagatgatcttgacagtcttattaatgaaatattcgaggagcctgactttgacagaaaatcttttgtaagtcactaaatgtacagtaattatttaagatgcttagactcgtcttaaagttaagtcacagtagccatctgggtatgagaattctgaggagtacaggtaaagaaaGGATTCCACTTTTTAAAATGTGGAGGGATAAAATGTTAGAAACTCTGTGTGTATTTCTGTTAACTCCTCAGTGAGAGTAAGAATTTGAAAAGCTGTTATTAGAGAAGTCAGCTCACAGACCACATTGGTCACTTAGAGTTCGTGGTAGTGTCTAAGAACGTGCTCTAAAAG
This mutation is a 505 bp deletion beginning at Chromosome 4 position 10,879,214 bp and ending after 10,879,718 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 46964 | CAT TCC CCA GTC ATT CCA GT | Common | A | |||
| 46965 | GAG TGA CAG CAC AAG GAT GC | Wild type Reverse | A | |||
| 46966 | TTT CTA ACA TTT TAT CCC TCC ACA | Mutant Reverse | A | |||
| 46967 | Fluorophore-1 | TTG TGA CAT AGC TCC ATA CCT GAA | Quencher-1 | WT Probe | ||
| 46968 | Fluorophore-2 | CCC TGC AGG GAT TCC ACT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 46964 | 0.40 uM |
| 46965 | 0.40 uM |
| 46966 | 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.