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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
>chr7:46788654-46788765 112bp GTGGAAAATGCTGATTTGAGG GAACCGTGAGACACATCC
Mut= 107 bp
Wt= 112 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TAAACACTTGTAAGTGAGCAAATGATTGAAGTAGCTGTTTTGTCCCTGGTTTCCTCCACTGTAACTAGCACTGGAATAACCTGGTCAAGTTATGAAAATATGAACTGTTTAATAACTGGTTTTAACAAAGAAAAATTACACATTTATACAATTGAGCCTATTCAGGTTTTGGAGATTCAGCTAAAAGTTTAAAAGGGAAATGTTCTTTGTCTGGCATATTTCAGTATTCTAGGATTATAAAGGAGTGCTGTTACATTGTGTCccgggcaggggagagaaggaaggaagtgagcacagacctgcaatacttagttcaagtggctaggttatttttaagtgttgggaatgcagcctaggttctcgtgcatgccaggtaggtgtcctgtaaccgaggtgcatccccagtgcatagcagatctcacgattgtctcctcatgctcttagtgtaccagcatagctgtgtctcggaaatggttggcgttaggcagcacaggaggaggactcaatctcattcagaaagatggctggaagcaaaggctgtttctctctcaccgggtaagaccaaggacattcacccactcccccccaatgtaatcaattatttagagtaaaagaagaatgaaaacgaactattcttgtctgatgttctaggaaggggcaatctctcagattgcctgctgctctcatgatgatgattatgttgctgtagctaccaggtaagaaacccttaaagctgacatgtgtgcttgtcaggatgaccaaccatctcatctcacccagcactgtcctggtgtcagttctgaaagtcacatcctggggatccaagtctgctcagtgcccaagtcacctgggaggggtcgttggtcaccctggtgtttgtggaaaatgctgatttgaggcaacaacatctaaaaggtcttaagagagaaatggaagagtcgtcacctctcccTGAGGAGCTGGTACACGTCTGGATGTGTCTCACGGTTCTCTCAGGGCCTGCTTCTTTTCTCAATCTTCATGCTTAAGCCTGTGCTATCTCTAATCTTGCTGAGAGAGAGTGGGAGCAGAGGATGTGCAAAACATTTGAACTCTTAAGTTCTCCTATTTTATTGGTTTGATGACCAAAACAAGGAAGTGAATACTTTGACTTGTAACTCCTTCCCCTAAGAACAGGAGTTTATATTGTATTTAATATGGCTAATATTT
This mutation is a 690 bp deletion beginning at Chromosome 7 position 46,788,692 bp and ending after 46,789,381 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48001 | GTG GAA AAT GCT GAT TTG AGG | Wild type Forward | A | Wt F | ||
| 48002 | GAA CCG TGA GAC ACA TCC | Common | A | Common | ||
| 48003 | AGG GAA ATG TTC TTT GTC TGG | Mutant Forward | A | Mut F | ||
| 48004 | Fluorophore-1 | AGA GAA ATG GAA GAG TCG TCA CC | Quencher-1 | WT Probe | ||
| 48005 | Fluorophore-2 | AGT GCT GTT ACA TTG TGT CTG AGG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48001 | 0.40 uM |
| 48002 | 0.40 uM |
| 48003 | 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.