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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
>chr9:99640090+99640200 111bp CCTCTGACTTGTGCCCTTCA GCCCCATTTATACAGTCAGC
Mut = 123 bp
Wt = 111 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
ATCAGGATTTCTGTTTTCTTTCCCTTCCCACTCCCACCGTTCTTTCTGTCTTTCTGTGGTGTTGGGAATTGAACCTAGGGTCTCACCCACTCCCGACCCTTCAAATTCTTTTTTATTTCTGAGGCAGCCCATGGACCCTGTGGAGGCAGAGTTTCACTACAGGATATTTccacgctgtctgggggatctaatccagcccttccaattgcttttcttctgtgtagtgccacttgtgtgacaagaccttcatgaatgccacctttctccggggccacatccaacgcagacatgcaggcatggcagatgtcggtgagtccaggcttcattggggaactgctgggcaaggatttggtgtgtgaggcccatgcacatagacctgctcttagagcctcttagcgattccacatggtgccctgtgcagtcttgcctgtgtaggggacaagaggcatcagacaggtaggtttagttttagttttccatgaacagtggccagtacttagcctctgacttgtgcccttcactgtgtgtggagcaggcagtgactcataagggcctttagcctccctgtaGCCTCATCTGTAGCAATCCGAGGCTGACTGTATAAATGGGGCAAATGAGCAGTCAGACCAGTTCTCTGGCTGTTCTGTGTACGGAGCTTATCACGACTTAATCACTTTATTTAATTGTACCATTTCAGATTCCAAAAGGAGTAACAA
This mutation is a 390 bp deletion beginning at Chromosome 9 position 99,639,764 bp and ending after 99,640,153 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 49330 | GCC CCA TTT ATA CAG TCA GC | Common | A | |||
| 49331 | CCT CTG ACT TGT GCC CTT CA | Wild type Forward | A | |||
| 49332 | ACT CCC GAC CCT TCA AAT TC | Mutant Forward | A | |||
| 49333 | Fluorophore-1 | AGC AGG CAG TGA CTC ATA AGG G | Quencher-1 | WT Probe | ||
| 50768 | Fluorophore-2 | AGC CCA TGG ACC CTG TGG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 49330 | 0.40 uM |
| 49331 | 0.40 uM |
| 49332 | 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.