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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
>chr5:74196089+74196194 106bp TTTCTGAATTGCAGCTTTGG ATCCCAACAGGGTTGGTC
Mut= 123 bp
Wt= 106 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CCGGAACCCCCGAGCCTCTCTTCTCGTCGCGCTCTCCCGTTCTTTCCAGTTTCTGCCCGCGCGCCCTCCGACGGGGTACCCTCCTCTCCTTGCAGCGCGAGCCGTCTCCTGTCCTGACTTTCCTCCCCGCTCCGTGCAGCCGGGCCGTAGCGGCGATGCGCCTCATCCAGAACATGTGTACCATCGCAGAGTACCCCGCCCCAGGcagcaccgctgccgactgctgcctgggggctgcgggccgccggctcgttaagatcgccgtggtaggggccagcggcgtgggcaagaccggtgagtccatgcgaggaacccgcttggtggaggggcgggacgtgggagacccccgctcggactggaactgagagacggtgggcgttgcagtgggatcctctgcctcttcatgtctctgctttaacgaagagggtcactggggcccagagcgtcccgccgactttcgggccagagtagaactttggggctttgggcttttggcaggatctggggcggaatgggggagctagtgattgtaacccctccaggagttccgtaaggttaggaagaagtttgcctgctggaaggcttaggtgtggcccagctgggctccgcggaggggctgggcctcaagacccttcctgctggcagagctgtaattcactcctacctccttgtgtctggtagagggtcaaggagccctcggtggccggctgtgactttctttggaagagttgttttccaggtgggggtggggtggagaaccgtctaacttagtgggttaacagtctgttttcttttctgaattgcagctttggtggtccgattcctcactaaacgattcatcggggactacgaacgaaatgcaggtgaggaaacgtacggagaccaaccctgttgggatggccgccctcatttctcagtacctttaaggacacttccttcagagtgacagtgtttagcatcaagattctgctacttccaattctgcacgccccctccccctcccccttccccctccccccagctgccatctggcctcattccattcttggcgcattcatcattttatcaagtgcccgaaagtattccctgtggtatgttttaactatagggaccccagttggaagcaccgtttgtactccaaggccttgtcctttaaaacatgcctgcaaaagtcataaaaaaaaaaaatacccaaccaaacaaaaaacccagctgcaagctcctcccccaccctctccctctgctggctgcctttcaggggcgacctttgaaagcaaaggccaaagaagagttgtaagcctaaagttctttccttaacaaagacaaataagtagctgctatgattctagcagggggagagcaaattgcttaactaagtagactgtatggttaaattagtagacttgtattaagtaccagttggggatttagttgaaatgagccatataagctgtcccaagataagacctggtggccttctgctttgatatagagctgatcagaatcatggcacatattcatgctaagagggatgctaactgtgcatttactaaccgtgtttgcttatgaagctattgtgggcaaaatataactgttcagagaatgataatataaaataaaaacagaaaccctgcgtatactgagccagaaactttcctccgtggaactactctaaatatggaaactcagttaaaattagaccagcaagtttctaaaaacccacaaactttgacctacttttgataaaatttgataatctactagcaggggcttaagactgcacgtggcctttaaacaagatctgaattaagtgcattaactagaagtaaatcacatctttctcttgtgtgtatcggtgcaagtcgtggtacattaaaaacaaacaaacaaacaaacaaaaacccaaaccataatgtttcccgaggctgcacatgaaagaggaacacactctcctttctctgtcttcataggtaatctctacacaagacaagttcacatagaaggagaaacgctggctattcaggtccaggacactccgggcatccaggtgagacactgatgctgacagggccagagcagacaccggcggctgaggcactgctcacttgtttaagtgtaagggacacgagatgtgggcagtcaggtctagacggtacttgtggtttgacagttaaattgtacctgaccgcaggtcagccttggcccggttcagtttggggagctgggagaagaaggggagcccctcttgtaggaggggaatcaccggagcagggggaaccaaaccagcccttagaattggcagtacttccttcctttggtttttatttatgtgcctttggcatcaaaacccaaatattcaggcccagtctccaacacccggaatcacttttatgttgtggaaagtttctctgtagacaaaagagctctgctaggattttttccccccttccccgtcctgaactgggcagttatccaggtttaaatgcttccagctcctatcttccccagacttactgtctagccagttaatttcccattttgtgcaactacaaagtcgctctgcgtggagttctctgcaggtccattttccacttgagtgtggtgagctgccctgttatttattccctccaagcagctggatcaacatattgttaagaaagcccaagtgtgtggggaatagaacttgtttacaacccgtaacccgccatccatcgctatgaagggttcttttgaattgagggatgtgctgtggccagagctcagtgccgtgcccactgctagctgctcaccttgctctccctcttctcctcaggtgcacgagaatggcttgagttgcagcgagcagttgaatcgctgcattcgctgggcagatgccgtcgtcctcgtgttctccatcacggaccacaagagctacgaactcattagccagctccaccagcacgtgcagcagctgcacccgggcactcggctgcctgtggtgttggtggctaacaaagctgacctcctacacgtcaagcaggtggaccctcagcttggcctgcaactggccagcatgctcggctgctccttctatgaagtatctgtcagtgaaaattacaacgacgtctacaatgctttccatgtcctgtgcaaagaagtgagccccaagcagcagcccagcagcacccccgagaaacgcagaacttccctcatccccagacccaagtcgcccaacatgcaagacctgaagaggaggtttaagcaggctctgtctgccaaaGTGAGGACTGTCACGTCGGTCTGAGGCACGACTCCTGGAGGGGGTTTGGTCTTCTGGGCAGAAAGCCCGAGGTTCTTCTTAGATGGGACCATGGCAGGCTGACAGCAGTTATGGTTCCAGAAAGAGCAGTGGCAGAGGGGCCTGAGGGGCCTTTTGGAATTCTGCAACAAAGGAAGTGAGGACAGATGGACGGATGCGGGACTGATCTGCCAAGTCGTTTACGTGGGGATGGGGCATGCTTTTGTTCCTCACTGTCTTCTAAGTGATGGTCAGGTGCACCAAA
This mutation is a 3,054 bp. deletion of Chr5:74,195,502-74,198,555(GRCm38/mm10),
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 74852 | TTT CTG AAT TGC AGC TTT GG | Wild type Forward | A | |||
| 74853 | ATC CCA ACA GGG TTG GTC | Wild type Reverse | A | |||
| 74854 | Fluorophore-1 | CGT TTA GTG AGG AAT CGG AC | Quencher-1 | WT Probe | ||
| 74855 | CAT CCA GAA CAT GTG TAC CA | Mutant Forward | A | |||
| 74856 | TAA GAA GAA CCT CGG GCT TT | Mutant Reverse | A | |||
| 74857 | Fluorophore-2 | CTG CCC AGA AGA CCA AAC CC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 74852 | 0.40 uM |
| 74853 | 0.40 uM |
| 74855 | 0.40 uM |
| 74856 | 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.