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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
Sequence
Wt Sequence (deletion in lower case):
TCTCTTGGTCCCTTTTATTTCTATAAATGCTCTGTGTTTTAAATTTCAAAATTAAGTTTTCATGTGGTGCTACTATTTTTCTACTTACTATTCCTGGCATGACGGTTGGATGCGTTCTTCCTGACATTAAGCAGTGATCATATTGTGAACTgcactggcactattattcaccctgctgtagtctgctgcccggtgacctgcctgtgccctcttttgcagataaacgcggtacgtgcgatagttcccaataagagcaacaatgagatcatcctggtcttgcagcactttgacaactgtgtggacaaaacagtgcaagcgttcatggaaggtaacgccagttccctcagtgtagactctggaacctgagccgcagcgcagtctccggacggcgctgcatactgtggtcttctgttagctagggtactcctttcattcccaagctatagaaaactggaagagatgctaagaaaactcctttgctgtcagatatgttctccccctccccaaatcaagaaactgtgcagctgacagactgtgggatttgaatGGAATTGGATAAAGTAATTGAAGACGCTATTGCAAAGGAGGAAGTCAAGTGGCCCACGAACACAGACCAGTTCAAGACCCAGCATTGATTTCCAAGCTCCATTATGAGTTAGTTTGGTTCTGGCAGGGGACTCAAATGTTATGAGTTCAGTTTCCTTACATTTCTATATGCTAGATAGAAAAAAGTAACAGCCTTGTCCAACTCCTTAGATTTATAGAGTTAATAAGTGCTTCTTAAAATTCTTTCCATATAACTTGTTTGAAAAAACTTTACTCTGAAGCTGTTTTGCCTTTGAACAGAGTGGCCAGGGCTCCTTGATGCGTTTTCCTGAATCCGCAAGCATAGAAT
This mutation resulted in a 396 bp deletion beginning at Chromosome 15 position 99,173,334 bp and ending after 99,173,729 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44431 | TAT TCC TGG CAT GAC GGT TG | Common | A | |||
| 44432 | AGC AGA CTA CAG CAG GGT GA | Wild type Reverse | A | |||
| 44433 | CCT TTG CAA TAG CGT CTT CA | Mutant Reverse | A | |||
| 44434 | Fluorophore-1 | TGA ACT GCA CTG GCA CTA TTA TTC | Quencher-1 | WT Probe | ||
| 44435 | Fluorophore-2 | TGA ACT GGA ATT GGA TAA AGT AAT TG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44431 | 0.40 uM |
| 44432 | 0.40 uM |
| 44433 | 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.