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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
>chr11:34401957+34402046 90bp TCCATGGCCCAGCAAAGT GTGGTGCGGTTGTTTCACA
Mut = 99 bp
Wt = 90 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
CCAGGACAAGCGTGCGGTTCTTCTCAGTGAGGCTCCATGGCCCAGCAAAGTATgaaagtgaggcctgtgctcctaaagcgaagcagccttgagtctgtggagcttgtgaaacaaccgcaccaccgcaggagcaaatcgcagcaagtgcgattcaaggaagatggcaacattaagaatccaactggcgtgaccgaggtcaacacccaaacccccgaagacccaggtgtaatggggaaggcacaggcctccaggcaccatcatcctaccacctactcgctgtctttccccaggtcccacaaggcaggaggattccgcagcatctccatccagacctcccccagtctcaggaagcatttccccgttttcaaaaggaagaaactcacgactagcaagtccctggtggaaatgccaacagcatccccaagtgccatccaggtcaacggcaacctatctgaacaggatattgtgtcctctgaccttgccttcctgaggctggctcagcatcttgaggatgggcctcgcaggctcaaaatcccccacccatttctccctaggatgcccaaggtgcaaagcaatgggcctgtcagcttctgcttagaatctgggacttggatgtcctcagagaaagccacagctgctattcaggttccagatgatatttgtcacagtccaacctgggaagctagagagtcggctctcagcccagaaagctcagctgaagagagcaactctatacctgctttgatctccatgtgtccaggggatgggcaaagagtgatgacgtcagagctggaaagaatgcccccctgctcaaataccaatagcagtgccagtaatatgccgggcacagaaaaactcacacccgaattgcttttgcccaaagacaaccctgatgacaaagaccttggccttccatcatctcagtcaaagaagatgtgtgttccctcaccaccacggactcacagttcgcctgagccaggctcccgtagccagccagtccacctaggaagatcttctgattgcccagcctcaggtgacaatcaccaggacctggagtcactcagaagtagcagtgcatccaagtctgtccctgtgtgctgggaacatgtgacaaagctccccagccagtcagacaccccagagcttcagactggtgttggaagtgagcaactccctgcctcgattccaaggcaggagaacagagctcagagcagcagggagataggtgggagtaatcacagtcacctggcgcagggagagctctgtgacctccaaggccggctgcaatcagtagaggaatcgctgcactcaaaccaagagaaaattaaagtcctattgaatgtgattcaggatctggagaaagctcacgctctcactgaaGGGTAAGGTGAATTTCTTTATGTTGAAAAACCACTGAGCCGAGTTGTTTTCTGGGAAGGGAAAGAATGAGAAAGCAAGGTCTCCCACAAAAAGTTCTTGCTCTGTTGTTTACCTAGACGCTA
This mutation is a 1327 bp deletion beginning at Chromosome 11 position 34,401,977 bp and ending after 34,403,303 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 51552 | TCC ATG GCC CAG CAA AGT | Common | A | |||
| 51553 | GTG GTG CGG TTG TTT CAC A | Wild type Reverse | A | |||
| 51554 | CCT TGC TTT CTC ATT CTT TCC | Mutant Reverse | A | |||
| 51555 | Fluorophore-1 | CCT AAA GCG AAG CAG CCT TG | Quencher-1 | WT Probe | ||
| 51556 | Fluorophore-2 | ACT GAG CCG AGT TGT TTT CTG G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 51552 | 0.40 uM |
| 51553 | 0.40 uM |
| 51554 | 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.