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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
>chr9:108576501-108576611 111bp TGCTGATGTGGCTCTTTACC CCAAGCAGGTTCTGGACTCT
Mut= 111 bp
Wt= 111 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
AGGTCCGGCTGAAGTGGAGGCACGGGTTTGGTAGGATGCTGTGTCCTGGTATATTCTGAGTCTGTATGCTAAGGAGTGGCCTGCTCCACTCATGCGTCCTAGGATCAGGTGCGTGCGTGCGTGTGTGGTGCTGATGTGGCTCTTTACCTCTGTCTAGGCGAGGGGCctgatttactggtgtacaacttggaccttggtgggcatctccgaatggtgaagagagtccagaacctgcttggtcactttctcatccatgggttccgagtgcgaccagagcctaaaggagacctggactctgaggccatgatagctgtgtttgggagcaagggcctcaaagttgtgaaagtcagctggggtcaaagccatcttcgggagctctggcgctctggcctgtggaacatgtccgactggatctgggatgtccgctggatcgagggtaacgtagccgtggccttgggccacaactcggtggtactgtatgacccagtgatagggtgcatgctgcaggacgtcccctgcacagacaggtgtaccctgtcctcagcctgcctggttggtgacacctggaaggaactgaccatcgtggctggcgcggtttccaatgagctcctgatctggtacccagccactgctttaacagacaacaaacccgtggcccctgaccggcgggttagtggccatgtgggtgtcatctttagcatgtcatacctggaaagcaagggcctgctggcaactgcttcagaagaccgaagtgttcgtctctggaaggtgggggacctccgggtgcctgggggtcgggttcagaatattggccactgctttgggcacagtgcccgagtgtggcaggtgaagctcttagagaactatctcatcagtgcaggagaagactgtgtctgcttggtgtggagccacgaaggcgagatccttcaagcctttcggggccaccggggccgaggtatccgggccatagccactcacgagaggcaggcctgggtggtcactgggggagacgactcaggcattcgactctggcacctggcaggccgagggtacccaggcttgggcgtctcatccctgtccttcaaatctcctagccggccaggtgccctcaaggctgtgactctggctggttcctggcgagtcctggcagtgactgatgtggggtccctgtacctctacgaccttgaggtcaagtcctgggagcagctgctggaggacaatcgctttcggtcttactgcctgctagaggcagctcctgggcccgagggctttggactctgtgccttggccaacggggagggtcttgttaaggtggttcccatcaacacccccaccgctgccgtcgagcagaaactgttccaggggaaggtgcacagcctgagctgggcccttcgtggttacgaggagctgcttttgttagcatcgggccctggtggggtgatagcttgtttggagatctcagctgcacccactggcaaggctatctttgtcaaggaacgttgccggtacctccttcccccaagcaagcaacgatggcacacatgtagtgctttcctgcccccgggtgacttcctcgtctgtggggaccgccgtggctctgtgatgctattccctgtcagaccatgtctattcaaaaagcctggggccggaagcaaggctattactgcagctgaggcacctggagctggtagtgggagcggtgggtctgagagtgtcccaacaggaataggccccgtctctacactccattctctgcatgggaaacagggtgtgacctcagttacctgccatggtggctacctatacagcacagggcgggatagctcctacttccagctctttgtacatggcggccacctccagccggtcctaaggcagaaagcctgtcgaggcatgaactgggtagctgggcttcggatggtgcctgatggaagtatggtcatcttgggtttccatgccaacgagtttgtagtgtggagcccccggtcccatgagaagctgcatatcgtcaactgtgggggagggcaccgctcctgggccttttctgatactgaggcagccatggcctttacctacctgaaggatggtgaggtcatgctctatcgggctctaggtggctgcatccggcccaatgtgattctccgggagggtctgcatggccgtgaaatcacatgtgtaaagcgtgtgggcacagtgaccctgggccctgaatttgaggtacccaacttggagcatcctgactccctggagcctggcagtgaggggcctggtctgattgacatagtgataacaggcagtgaggacactactgtctgtgtcctagcacttcccaccaccacaggctcagcccacgccctcacttctgtctgtaaccatatctcctctgtgcgagccctggcagtgtgggctgttggcaccccaggtggcccacaggatactcgaccagggctcactgctcaggtagtgtctgcagggggccgagcggagattcactgcttcagcgtcatggtcactccggacgctagcacccccagccgccttgcctgtcatgtcatgcacctttcatcccaccggctggatgagtactgggaccggcagcggaacaagcacaagatgatcaaggtggaccctgagacCAGGTAAGGCGGCCTCCCTGTCATGGGCGGGGCGTGAGAGGGTTATAGGAAAGTCCTGACTCCTGACTGCAGGCCCCACCTGACCGGTGTGTACCCCCTACAGGTACATGTCTCTTGCTATTTGTGAGCTTGACAACGATAGGCCTGGCCTCGGCCCTGGCCCCCTTGTGG
This mutation is a 2,470 bp deletion beginning at Chromosome 9 position 108,574,104 bp and ending after 108,576,573 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 42316 | TGC TGA TGT GGC TCT TTA CC | Common | A | |||
| 42317 | CCA AGC AGG TTC TGG ACT CT | Wild type Reverse | A | |||
| 42318 | CCT GCA GTC AGG AGT CAG GA | Mutant Reverse | A | |||
| 42319 | Fluorophore-1 | CTT GGA CCT TGG TGG GC | Quencher-1 | WT Probe | ||
| 42320 | Fluorophore-2 | AGG GGC CAG GTA AGG C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 42316 | 0.40 uM |
| 42317 | 0.40 uM |
| 42318 | 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.