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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
>chr9:45900465-45900569 105bp GCACTTAGTATAGGACACACTCAAAAC TTCTCCACCATCAGAACAAACTTCT
Mut= 90 bp
Wt= 105 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
ATCTAAATATTTATGAAAATATGCATAACTAAGTTAGTATATGTAACTACCGTAACTAACATTGTGCTATTGTTTCCTATTCACAAAAGCTGTTGGCAGTGGGCATTTATTTTTAGATCTTGAGGGTCACTTGTTTTTAAGCACTTAGTATAGGACACACTCAAAACTTTTGCAAGGTCTCACTGACCAGGTTAgaatacctacaaattgttgggtagaaagaagtttgttctgatggtggagaaggcctatagcatctaggtttgttctagtggctgtgttggggatgtggctcactagtagagcactgtccagcatgggtttgatccccagcactggtgctagtagcagaatcctgggtctttggtgtgctagtcaggtgacataccactgagctacaacctcatcccagattttttttttttttttttttttttttttttttttggatagccccaaagactctgcccagaaacagaagaacttgtctccgccgagtgtaagtagccaaatgataaccaaggagagtaacagaaatgcccatttggaacatccagaacagaatcctggttcatcagtgggtgatacttcagcagcgcacgaggaagttgtaggagaaaacttggtagccacagccctttgtctttctggcaatgggtctcagtctgatttgaaggacttgaccaacccagcaggagaggagggggacacgagcctccgggagagcctccatccagtcactcggtctcttaaggcagggtgccactcaaaacaacttgcctccgggaattgctctgaagagaaatgcccagcagcctccgtcctaaaggaaggtagcagggatgcaggcttggacttgctacctgtagtaccgcctgcaaatggggttgaaggagtgagagtggatcaggatgatgatcaggatagctcttccttgaagctttctcagaacattgctgtgcaggtcaagtatcacatttctactactaaaatggaattggggacgggaggggagaagccatttaggtggtagagaactgcattgtgtcattttttgctagtagtcatgacacagtttgcatatagtatcatgtaatggcttcattctgctattgcagaaacaagaaagccagtatctaattgtataatatgcaggaattattatgacctatgtgtaacctctttgGATGAAAACTTGATTGGTGTAAATCCTTCCTGTTCTTTCCTTTTATTTTTTTCTTTTTCTTTTCCTTTTCCTTTTTCCTTTTTCCTTTTTCCTTTTTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTTCTCTTCTCT
This mutation is a 992 bp deletion beginning at Chromosome 9 position 45,899,524 bp and ending after 45,900,515 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 40632 | GCA CTT AGT ATA GGA CAC ACT CAA AAC | Common | A | |||
| 40633 | TTC TCC ACC ATC AGA ACA AAC TTC T | Wild type Reverse | A | |||
| 40634 | AGA ACA GGA AGG ATT TAC ACC A | Mutant Reverse | A | |||
| 40635 | Fluorophore-1 | AAT ACC TAC AAA TTG TTG GGT AGA A | Quencher-1 | WT Probe | ||
| 40636 | Fluorophore-2 | CAC TGA CCA GGT TAG ATG AAA ACT T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 40632 | 0.40 uM |
| 40633 | 0.40 uM |
| 40634 | 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.