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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
>chr9:37517492+37517638 147bp GCATGTGTGTGTGGCTACCT AGCATGACTTGGCAATTTGA
Mut= 147 bp
Wt= 145 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
ATTTTATTGATTTGATTGCTCCTTCAGTTGTAGACATTTTTACCGTGACAGAGTAAGTTAGGAAGGAACAGACTCTGTAAGTGACCTGTTTTGTTAGCTACTTCGTCTAGGCAGCTGTTTTTCCCCTGTACTTAACTAAGAGTAAGGAAGATGTTTTCCACTTGGGGAAATTCTTCTTTCTGTCCTCTTTCCCTCACATTTTCCAAATTCTGGGCACCATAGCAGCTCTCTCTCCTGTGGCAGGTGTACATCCTATTGGCTGGCTGGTATTTCTTTTTTTCCTCCCCCTTATCCTTTTTAAATGGGGGAGGGGGCATAAAATATgtgtattggatacatgcaataatgttgtaatgtttcttgttgttttatataattttataattgtaaattaattgtttgaattataacatgtttgagtaaatgctaaattagtatttttctaatatgataatgcatttgaaatagtattcctgtaacaatgtgtctgtgtttgtctgtctgtgtctgtctgatagtaattaatatctgtggtccgtatctggaggaggaagtacagctttaaaggaataacaaaagactttgtgtcttagacccttcgcaggtgttacagtcgggtgaaagaacatggtgttgggaaaagaaagagcagttacacatttgaacagttggaacaggtgtttggtcagggaggatgggatgctcagccctgccagcctgtacttattaacagtagtggcttgtaccaggagctggagtcagatggcagcactatggaggactattcacaggaggactggggaaaccacagtcaggagctccatggctatccaacagatcaggaattgggtaagaaaacaacatgtgtgcatgtgtgtgtggctacctgatagtgcaatgaatggtatttataccatctgtaatggcttctagtgttgtcagcacaaggcatttgccaaactgtttcagtcaacatggacatccttgatttaacatcaaattgccaagtcatgctcactattagctacgtggtgacttgcatactgttaagttccttgaagatagagacttcctaatctgagtcatacaggagGTTCACTGACACAATCTTATGCAATTCAGAATACCCCTGTCTGTTCTTTGTAGCACTAAGATTTCCTGCTGAGAGCACTGACTCTTGGCCCAACTTCTTTAAATCCACACTGGTCCAATTAAGAGGCTACATGTAGTTTTTGTTTGCTTGTATCCCTGTATGTCAAGTGCTCCAGTTTCTTATTTGGGAAGTATTGATCAGGTTTTAGCTATAGCCTGTGTACATCAGCAGCAAGTTGTGTAATGAGTTTTAGTAGGAAATGCCCTTCCGCCTAGTGAAGTGCTCAGGTGCCGCTGAGAAAGTTCAGGCAGGATTGGCAGTGTAAATTTGCTTCCCTTGCTTAAGGGAAAAAAATACAGAGA
This mutation is a 769 bp deletion beginning at Chromosome 9 position 37,516,948 bp and ending after 37,517,716 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 49361 | ATC CTA TTG GCT GGC TGG T | Mutant Forward | A | |||
| 50035 | GCA TGT GTG TGT GGC TAC CT | Wild type Forward | A | |||
| 50036 | AGC ATG ACT TGG CAA TTT GA | Wild type Reverse | A | |||
| 50037 | AGC AGG AAA TCT TAG TGC TAC A | Mutant Reverse | A | |||
| 50038 | Fluorophore-1 | AAT GGC TTC TAG TGT TGT CAG CA | Quencher-1 | WT Probe | ||
| 50039 | Fluorophore-2 | ATG TTC ACT GAC ACA ATC TTA TGC AA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 49361 | 0.40 uM |
| 50035 | 0.40 uM |
| 50036 | 0.40 uM |
| 50037 | 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.