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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
>chr1:191070326+191070441 116bp CCAATGAAGCAGGTCTGG AGGCAAAGACAATGGCACA
Mut= 121 bp
Wt= 116 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletions in lower case):
TTATTTTTTTTTTAAACTGCATCTGTCTGCAGTTACTTCTTCCAGGGTGCTTCATTTCTGTGCTCTGTCCTAGACCTTCTGAGTCCCGATTTACACACTAGTAAGGTTCCTGGGCAATCACTCCTGCTCGAGCTTGAGAACCACTGTCTACAGCCGAGGAAAGAGATGATAAAATAGTTGCCACTGAAGTCTGAGGAAGTCCCCCAAGTCACCATGCCTTTCCTTTCCCTGCCCTGGTGAGTCAGGTCCTAGAGTGGGTGCCCTTCTCACTGCTGTCACAGGTTGGGTTGCACAAAAATAAAGTGAACCCTGTCCATGGTATGAGATGCCTTTTATAGTTTAATTATGGTGGGGAAGGGCAGAGGACTGGCCCAGGGCCCTGCCCATGCGAGGCCCATGCTCTGGCACTGACTGCATCTCTGCCCTCCTCTCTGTCACCTGTCCTATGCTGTTTGGGTCGATCCGTTTGCAAGGCTCAGTGCCGTCAGTTTCCTCTCCCCGAGGTCCTTCTCCCTGAGCGATCTCATCCAGTGCTTTGTCCGCACCTGGATTTCAAGCCTAAGTGCTGTTCTTTATCTCCCACTGCTCCTGCTGCCGTCTTCTGCAGCCTTCGTCTCTGTGGCCTGAGGCACTTATCAGGTGCCTCTTTCTTGGGACTATGCTGCTCGCCTCGTGTTTCCTGGCGCTTGCAGATTAATCAGTGCGgagatgaggggggctccgttaacctgccatgtttgttttctttattctgtgacttatgcttatataatatttctatactaataatggacttctctgaaatagattctgacatcaaagtgcttgaagatgaatttgatgagctcatagtagatgtggcaaccaaacggaggcagtatcccagaaggatccttgagagtgtcatcaaaaccttgaaagcacagcacgcgagtctggtgagacgcttatatctaaatccctacacagtattttaccacagtagagagttagtttacttacctttcatatagtgcaggctggtcttgaacacctgatcttccttctctcactggctggggtcagtcaccatacctatcacatatcagtcttctcagaattattgaaactggagtaaatgcaaatgagccctgaatcataatgccaatgaagcaggtctggcagcatagacctgaggcaaatgctgagGAGATTGGCTGGGTGCCATTGTCCCGAGAAGTGACCACAGCAGGCTTTAGATTGTGCCATTGTCTTTGCCTTTAGTTTTTTAAGTCAGCAGACTTTTAAGTGTGACCTTCCAGTATTGTCTCCTTCAGTAATGCTTTGGGGCATTAAAGGCCTGCACTTGACCTTAGAGTCCTGCTGTGACAGGAAAAAGAAGTCCTTGAGGATGTTCACTTTCTCACACATTCGGCCTTAAAGAGCCCTTCCCACACTGCAGAGCAGAGAAAGCGTTATCTGCCGTTGACTGTGTCATGCCTCCCTCTAACTTACCTCCTTGACCTGCTAGAGAAACTCTGGAACTAGTCAGCTCGAGGAGGGGGGACAGGCCTGGAGAGCATGAGTGTGCCCTGTCACTGACTGTGGCAGGTCCAGCTGCTCCTGTGTGCTTCCGTGAGGAAGACGGAAGACTGCTGTGGTAGTGTAGCCACTTTTTTGAAGTTTCTGTGGAAATAGGAGTTGTGAGTTCCAGATATTTGTCCTGAATGAGCATCCGCCACACTGCGGTCCTCGCCCGGTATTTTAGAGCTGCCTTCCCTGTTTCCTCTGACTGCGATCTTCATAGTGCTGTTTGTTACTCTGCAGCAGAGGGCGGTGCATAAAAGGTATTCAAGCCTAAAGACATTTGCCACCCAGAGTTCAGGAGCTAAAAAGGGTTAAACCTGGAGGCCAGCACTTTTGACTGTGTGTCTGCTCATACCAGCTGAAAGAGAGGCTGAGTCATCTGAGGCATTACGTGTCTAAGGTAAATGTTTTAATTTGTCCTGTGTTTAGAAGCAGTATCACCCTGTTGTACATCCTCTGGACCTGAAATGTGACCCTGACCCAGGTGAGCCAGTGTGA
This mutation is a 483 bp deletion beginning at Chromosome 1 position 191,069,888 bp and ending after 191,070,370 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48414 | AGG CAA AGA CAA TGG CAC A | Common | A | |||
| 48415 | CCA ATG AAG CAG GTC TGG | Wild type Forward | A | |||
| 48416 | ACT ATG CTG CTC GCC TCG T | Mutant Forward | A | |||
| 48417 | Fluorophore-1 | AGA CCT GAG GCA AAT GCT GA | Quencher-1 | WT Probe | ||
| 48418 | Fluorophore-2 | ATC AGT GCG GAG ATT GGC T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48414 | 0.40 uM |
| 48415 | 0.40 uM |
| 48416 | 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.