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Notes
Taqman Probe/Endpoint protocols use a different analysis than qPCR. Please see your equipment manual for more information about Taqman endpoint analysis setup.
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
>chr8:40826880+40826996 117bp TTCCTCCAGTTTCCATGTTC ATGAGGTATTTCATGCTGAA
Mut= 84 bp
Wt= 117 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TAGAGTCATGCCTTTGAACAAACCTCAGCCATTTAAGCTCACATCTTGTTCTTTCTCTTCCACTGTGGCCTGCCCATACTGAAATACTCCAATTTCTGTCTTTTCATTTAGTCCTGCTACTCTAATGCATCAACTCAGTCAGAAGACAGACCATTAAAAGCCAGCACTGCATGCAGACAACACAAAGGGCATCCTCCTTTGCTGCCACAGAGGACAACATAGCTATGGATAAGGCTGTGGTATACACAAGGATACCTCATCTGTACctctggctagagatattgaatattctttcatcatggctcttgactgggtatgctcagcacaccagcctcccagaagtagtgatacccttacgggtcacaggcaacagaccaatgtgggctatgggatggctgacctatagcctgcattttgggggccagaaacattttattcacataaaggccaagaagttcttggtttctagactcttctctgtattcacctacactaagcaaggtgctctgcacaaggaccagccttatgtccagaatgattgctactaccatggccatatggatggagacccagagtctatggttgctattaccacctgttatggaggctttcaaggaatattacagataaatggcacagtctatgaaatcaagcccaagaacctttcttccacatttgaacatttggttcacaagatggacagtgaggagacagaattataccccatgagatgtgcattgacagaagagatagcaaagcaaatgaaacttcagcagaatgagaaccccactctgatgcaaagccactatgaaggatggtggacccacaggagttttcttgacctggcattggttgttgactgtgagcgagttcattatcataacaataacacatcacatgtcttagtggaagtattcaccattgtcaatataataaataatatttatgagacactggatgttgagctagttttacttggagttgagatgtggaatgaaaaaaaccacgtgcagggacgtaacataaacgaattcctagttgaattttgtgattggaaggctcgcagccttaattttcgaattccaaatgacattgcacatatttttgtgaatcttgattttcaaatttaccttggtgtggcctatgttggaggtgtctgtttgccatcttataactgtggagttgattgtctgttaggaggtaatcttttttattttggacatattatagcacatgagattggtcataatctgggtatggagcatgatagcagctcatgtacatgtggaagaaatgtttgcttaatggctccagcagataatggtatctcgaagttcagtaactgtagttattcttacttctggactaagtaccctactgccaagtgtatgcacaaggaaaagaagtcaataggcatcctccgaggcaagctctgtggggatggagtggttgacgatggagaacagtgtgattgtggatctgcaaaaaactgtgcagacgatccatgttgtaagcgcagctgcaccctcaaagatggcgctgcctgtgcttttgggctttgctgcaaatattgccagatcatggaggcaggcacagtttgccgaaaaagggacaatgaatgtgatcttccagaatggtgcaacggacattcacataagtgtcctaatgacgtttatttgcttgatgggagtccctgtagagatggtggctactgctatgagaagagatgtaataatcgagataaacagtgtaagcaaatcttcggcaaagaagccaggagtgcagatcacagttgctacagagaactcaacacccaaggtgaccgttttggcaactgtggtatgatcagagatgcatacttaagatgtcatgacccagacatcctctgtggcagagttcaatgtgagaacgtaacacgcattccatttttgagggaccattccactgtacactggactcacctcaatggtgtcacctgttgggggactgactaccattttgggatgacaatccctgacattggtattgtgaaagatggcacagactgtggtccagagcatgtgtgtattaataagaagtgtgtcagtaagccaatttggacaagtcagtgttcacccactacatgcagcatgaaaggtgtgtgcaacaatttacaccactgccactgcaaccgggggtgggacccaccacattgcctgaaaagtggccttggagggagtattgacagtggccctccgaactataatgaaaagaaacacaagaaaagtattgggctagtgagtcttttttggattctttttgcttgtttttccatattgttcattctgttcctttttttcttgaggagttatatagaactacctatgagtgaagaacccaaagttcccaccccagaaaagaaagaagatggcaatgatgtaatgaacactgaaaaagaataaaaacaatttgtaatttcaaatgtcattattaaatcttcctccagtttccatgttcaatatgtgttatcttggtatgtgaaacactatgtgtttctgaattcaatgacacaaagtcCTAGGGATATTTAGTATTTCAGCATGAAATACCTCATCATTCAATAAAGCTATGATCATAGTACAATCATAACTCACGATCTTCGCTCTCAGCCGCCTTAATGCTTCATGTTCTTTTACAGTGTTGTCAGTGAAGGCCCACCCTAGGGATCCTATAAGCCTGTGGTTTTGATTTTGCCTCTCTCAGTTCCAAACCAGCTCCTTATTATTTTGCTTATCACATGGGATATGGGCTTCTTTGAGTCCTGTTTGCTCACTGGTGGACCTCCCTA
This mutation is a 2,290 bp deletion of Chr8:40,824,670-40,826,959 (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 77374 | TTC CTC CAG TTT CCA TGT TC | Wild type Forward | A | |||
| 77375 | ATG AGG TAT TTC ATG CTG AA | Common | A | |||
| 77376 | Fluorophore-1 | TGT GTC ATT GAA TTC AGA AAC AC | Quencher-1 | WT Probe | ||
| 77377 | TAG CTA TGG ATA AGG CTG TG | Mutant Forward | A | |||
| 77378 | Fluorophore-2 | AGG ATA CCT CAT CTG TAC CTA GGG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 77374 | 0.40 uM |
| 77375 | 0.40 uM |
| 77377 | 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.