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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
>chr2:120760071-120760188 118bp TGCACAGCTCACAGAGTGGT GTAACTCAAAAGCAGAAGTGAGG
Mut= 120 bp
Wt= 118 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case; insertion with carrots ^g^ (CCCC insertion):
TCTGAGTTCGAGGCCAGCCTGATCTACAGAGTAAGTTCCAGAACAGCCAGGGTTATACAGAGAAACCTGTCTTGGGGAGAAAAAAAAAAAGAAGAAGAAGAGGAGGAAGAGAAGGAGGAGGAGAAGAAGAAGAAGAAGAAAAGAAGAAGATGTCCTCTTTATAAATATATAAATGGCCAAATAAGTGGTTTACGTGAATAGATATGAGCATATAGACACACACACATATGCATGTATGTGTATATATATGATGTTTTCTTCTTTGA^gccatggattctgaaggaaccgtcattataaatctttttaaaggatcttctttagaggaacgtgcctggattgctcttggctcactctagttttttttcctttgtggtttagcttctaacatccgtgtttgacaatagcatcaagacctttggagtaattgagagtgacccgtttgactgggagaagagtggaactgatggctccctgacaaccaccaccacctctgccacccctcagctgcacacccgcctgacccctgctgctatcgggtatgtcctgcacagctcacagagtggtacttttctaaatgcaagaacttccttagtctactctgaatccctcaatttaaacattccattgatttact^CTTTCCCTCACTTCTGCTTTTGAGTTACTTAATTCAGAAAACACCTTCTGTGAAATGGGAGTTTAGAGCTAATATGACTTGTTGCTCTCACAGTACAGTAGGTAGAGGGAACAACTTTTTTATTTCTAATATATATATATATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATTATATAATATATATTTTATATGTATGAATATGGAAAGAATGATTTATAATTATTCTTTTTATTTTTATGTTTAATTATGTTG
This mutation is a 368 bp deletion beginning at Chromosome 2 position 120,760,099 bp and ending after 120,760,466 bp (GRCm38/mm10). In addition, there is a 4 bp (CCCC) insertion at the deletion site.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 47968 | TGC ACA GCT CAC AGA GTG GT | Wild type Forward | A | |||
| 48094 | GTA ACT CAA AAG CAG AAG TGA GG | Common | A | |||
| 48095 | GGC CAA ATA AGT GGT TTA CG | Mutant Forward | A | |||
| 48096 | Fluorophore-1 | CCT TAG TCT ACT CTG AAT CCC TCA ATT | Quencher-1 | WT Probe | ||
| 48097 | Fluorophore-2 | AGC ATA TAG ACA CAC ACA CAT ATG CA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 47968 | 0.40 uM |
| 48094 | 0.40 uM |
| 48095 | 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.