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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
>chr17:46890543+46890692 150bp CAACGCGAAGAAACCAATAG CATCTTGACTCCAGGCTTCC
Mut= 98 bp
Wt= 150 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GCAGCTGCCGCTGCTGCAACTCACCCCACAATCCCGGATATAAGTCCTGCCCCTTTAAGAAGAAAAAAAAAAAAAAAAAAAAAGCTTCACCCGACTGCCTCCCACCCCACCCATCCACCAGGACCTTTGTCTCCCCACCCCCTCCTGTCAGCCGGCCCCGCCCTCCTTCCCGCCCCTCCAACGCGAAGAAACCAATAGGCGAGGAGaatatggagggagctgacgtcaccgcagctggccaatcacagcgcgcgttggtgggaggcctcgcgcggagagcgagcgctcggaggcaaggcggcgggggaggaagcctggagtcaagatggagggtgtggactgctccatggctctggccgacgctgccgctggctccccgcgggacctgagcctggtgcccgagcgacttcagaggcgcgaacaggagaggcagatagaggtggagagacgcaagcagaagcggcaggaccaggaggtggaggaggagaagagcggcttcttcgccgccgccttcgcccgggagcgcgcagcggtcgaggagctgctgcggggcgaggcgtcggcggagcggctggaagaggcggccaaccggctccagggcctgcggaagctcctgaacgactcggtgctcttcctcgccgcctacgacctgcggcagggccaggcggcgttggcgcagctgcaggctgtcctaacggaacgtcgccaggagttgcagcccaagaagcgcttcgccttcaaggcacggaagaaggatgctgcggggaccgcccaggtagacgccgcacccgtcgcctcggcggccccttctccaccggtgaccaaggaggaggagggagctcccggggccagctgggcctgtggcttctccaacctggagtcccaagacctggagaagagagctgaagagctgcaccagcgcgacgtccttttgagcgacctgaccaactgcactgtcaaactgtgtggcaatcccaacaccttgcggcttgcgaaggcccgcggctgcaaggtgctctgcggcccagtgaccacctctgtgtttctggaggattgtcgagattgcgtcctggctgtggcttgccagcagctccgtgtgcacactaccaaagacacgcgcgtcttcttgcaagtgaccagcagggctatcgtggaggactgcagcggcatccagtttgccccttacacctggagctacccggggatcgacaaggacttccaggactccggtttagataggagcaaaaataactgggaccaggttgatgatttcaactggctggcccgaaacgtggcctccccaaactggagtattcttcctgaagaggagagagacatccagtgggattgagcactggccacactcagtgtgatgtcccattcggtttacctgatattgtcttatgtttgcggtgataggggttagaaattgttgaaactcccctcctgtgagtcctattaaattcatagcagtgtaacagacttttgctggtttggccttcccctgcctccctttgagTATGACAGCGTGAAGAATTAGGGGGAGAATGGAGGAGCTTTGAGGGATTTGCTGTTCTTACCGTTGATCACAGGGAGTGATAAAGTGGGTCTTCAATAAATAATAAGGATGTCCGGAGAATTCCATTAGCGGACGGTTTAAATGGAAGAAGTAGGCCCATAAAAGCTCCATTAACGGCTGTTTTCCTCCCACTAAAGGTTTGTTCTGTGTCT
This mutation is a 1313 bp deletion beginning at Chromosome 17 position 46,890,571 bp and ending after 46,891,883 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 50196 | CAT CTT GAC TCC AGG CTT CC | Wild type Reverse | A | |||
| 52533 | CAA CGC GAA GAA ACC AAT AG | Common | A | |||
| 52534 | TGA TCA ACG GTA AGA ACA GCA | Mutant Reverse | A | |||
| 52535 | Fluorophore-1 | AGC TGA CGT CAC CGC AGC T | Quencher-1 | WT Probe | ||
| 52536 | Fluorophore-2 | CGA GGA GTA TGA CAG CGT GA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 50196 | 0.40 uM |
| 52533 | 0.40 uM |
| 52534 | 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.