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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
>chr12:81510760-81510896 137bp GCCGTATCAAAGAGGATGG AGGCCAAGATGGAATCAGA
Mut= 124 bp
Wt= 137 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
AATATCACCAACAGGATCAAAATCAAGTGTGGGCAGAAGCACTTATATCAAGACTCAACCATACTATTTCCCATGTTATATATATGTACATATATATTTATATTTCATCTAGCCAGTCATTTTGATCACAGTTGTGATCACAGTACCTGTAgtactgcctggttcgaattagcaatctaggtacttttgaactttgttcataatgacttattgactttgcccatgactgtgttttagcttacctcacagtctgaattattagtctctcttccccactagaccatttgcctctcctttcttctcctctaatctttctttcttcttcgtccctctctcctcactcttccatgattttctcagggctgggcttcaacatcgtcggtgggacagatcaacagtatgtctccaacgacagtggcatctacgtcagccgtatcaaagaggatggggctgcggcccaggatgggcggctccaggagggtgataagatcctctcggtgagaaatggcttcggctttctttgctatactttgctccctagaCCACATCTGATTCCATCTTGGCCTGCCTGTTTGGAAATGAAAATATTTAGAGTGTATCATTGAATAGGAAGAACATTGATCAGTCTTCCAACTTTACCGCTGGGATGGGAGGATAGTATTATTCTTGTTTGACAGACAAGGCAAATTTA
This mutation is a 391 bp deletion beginning at Chromosome 12 position 81,510,784 bp and ending after 81,511,174 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 40033 | GCC GTA TCA AAG AGG ATG G | Wild type Forward | A | |||
| 40034 | AGG CCA AGA TGG AAT CAG A | Common | A | |||
| 40035 | GAC TCA ACC ATA CTA TTT CCC ATG T | Mutant Forward | A | |||
| 40036 | Fluorophore-1 | TCG GCT TTC TTT GCT ATA CTT TG | Quencher-1 | WT Probe | ||
| 40037 | Fluorophore-2 | TGT GAT CAC AGT ACC TGT ACC ACA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 40033 | 0.40 uM |
| 40034 | 0.40 uM |
| 40035 | 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.