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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
>chr9:95493701-95493827 127bp ACGTATTTAGGAGGATGAGAAGG ACCTTTAGCTGCATTAACAACC
Mut= 148 bp
Wt= 127 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
AGGTTTGTGTATTTTGTTTCTTTTTCTTTCTGTATATGGGTGTTTTTGTCTGCTTGAATGTGTGTGCCTGGTATCCAGAGAGGTCAGAAGAGGGGGTTGGTCCCTTAGGACTGGAATTAGAGGTGGTTGTGAGCTGCAGTGTGCGTGCTGGGTCTTGGGTTCTTAGGGAGAAGAGCCAGTGCTCATTACTGGTGAGCTTCCACTCTCTGTGTTGGCATTTGCCTTTAAGACAGTATTTGGCAGTCAGATCTCTGTTCAAGGCCAGCCTGTCTGGATACTGAGTTCAAGTCTGCCTGGCCTCCACAGTGAAATCTTCTCTCAAAACAAAGCAAACTGAATCAGTCAAACCATACCCAAACAgcagttcttgtgtaactaatatttcgttacgtatttaggaggatgagaaggcagcggccgaaatttatgaggaatttcttgctgcttttgaaggaagtgatggtaataaagtgaaaacgtttgttcgaggcggggttgttaatgcagctaaaggtaagtaccaatatctggctttgaaaacaacccttgGATAATGTGATAACAGATTAATTATGGAAGACATTTGACTTTGAATTATCACTTTTCTTTTCTATTAGCACCCTCTAGTGGTTAGATTAATAAACAATATAAATGATCAAATATGAAGTAATTAAGACTCAAATTTGATTAGTGGGACTGTATGAAATAGATAATAATAATAGTAATAATGATGATGATAATAATAATAACTAGTACCTTTCAAGTTTAAAAAAAGTGAATTATGTTACAGAATTTTTAATGTTTTATAGTTTTGCTATAAAGTGGTCAAATTGTGATTTTATTTTTTCAGGTTGAGTGGTAGTATGTAAATTTAATTTTATATTCAAATCTTAATACTAAAATA
This mutation is a 189 bp deletion beginning at Chromosome 9 position 95,493,667 bp and ending after 95,493,855 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 47650 | CCA CAG TGA AAT CTT CTC TCA | Mutant Forward | A | |||
| 47652 | AAT CTA ACC ACT AGA GGG TGC T | Mutant Reverse | A | |||
| 49283 | ACG TAT TTA GGA GGA TGA GAA GG | Wild type Forward | A | |||
| 49284 | ACC TTT AGC TGC ATT AAC AAC C | Wild type Reverse | A | |||
| 49285 | Fluorophore-1 | CGG CCG AAA TTT ATG AGG AA | Quencher-1 | WT Probe | ||
| 49286 | Fluorophore-2 | ACC CAA ACA GAT AAT GTG ATA ACA GA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 47650 | 0.40 uM |
| 47652 | 0.40 uM |
| 49283 | 0.40 uM |
| 49284 | 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.