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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
>chr12:81485273-81485363 91bp AGTTACAACTGATGAGCCACGA CAGATAAAGGGCGGGGTTG
Mut= 91 bp
Wt= 91 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
AGTAGGATAGAGGGTTTGCGGAGCAGAAACCGGGACGGGGGATAGCATTTGAATTGTAAATAAATAAAATAATAAAAAATTTTAAAAAGTTCAGTTACAACTGATGAGCCACGACTTAAAATGTGTTCATTAACCTGGTGctacttgtgcattagcttaaaaaccaaccccgccctttatctgagtttcttgagtcgcagaagtcaagatccagaatatgcgaattacagattttccaaatcgaaaaacccaattgcacccggcgtatcctgaggtcacggaaccagagctcagaagcctcgacttccgggggagggaggaagcagaagtgcgttaaggccaatcgtttccgtttgcctcggggaggcttatcaggcgtggacctggcacccaaagcctgcgctgagtctacaacttcgcctgagacgtgttgcgggccgcgctgggctcgctctggaagcggctgactaagcaggccgcaccttctgccatgattgcgcccgctgtgttgcgagctctgcgtaagaacaagactctacgctatggagttcccatgttggtgagcggctgtgacgggagcaggggctgcgagtccggaaggggcgggccctctttggctgagtggcgtattggtgctgtctttcggatactgctgactagggggtctgtgggagccaccggagctttgaggtttttgtttttgtttttaagttcacgctcaaaagtgtacctcgggtcccgccatcccccaccccccgtttcctttcccttcgtgaacgcgcgtgTTACTATCCCCGTTTTACAGTACCTTTCCTGGGTTAAATGTTGAGATTTTGCAAGAAGACTAATCAAATGTCTTCTTTAATAATTACATTCATTTGTAGTAATTATGTTCATAATTTGTTGTTTG
This mutation is a 645 bp deletion beginning at Chromosome 12 position 81,484,671 bp and ending after 81,485,315 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 45359 | AGT TAC AAC TGA TGA GCC ACG A | Common | A | |||
| 45360 | CAG ATA AAG GGC GGG GTT G | Wild type Reverse | A | |||
| 45361 | CAA CAT TTA ACC CAG GAA AGG | Mutant Reverse | A | |||
| 45362 | Fluorophore-1 | CTA CTT GTG CAT TAG CTT AAA AAC | Quencher-1 | WT Probe | ||
| 45363 | Fluorophore-2 | AAC CTG GTG TTA CTA TCC CC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 45359 | 0.40 uM |
| 45360 | 0.40 uM |
| 45361 | 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.